Methods for enzymatic decolorization of chlorophyll

ABSTRACT

The invention provides the invention provides compositions and methods for the enzymatic treatment (“bleaching” or “de-colorizing”) of chlorophyll-comprising compositions, e.g., algae preparations, chlorophyll-containing or chlorophyll-contaminated feeds, foods or oils, for example, vegetable oils, including oils processed from oilseeds, such as canola (rapeseed) oil or soybean oil, or oil fruits, such as palm oil. In one aspect, the invention provides methods using a chlorophyllase enzyme for the enzymatic hydrolysis of chlorophyll in an algae, an animal (e.g., a fish) or plant preparation, a food or an oil. In one aspect, the chlorophyllase is immobilized onto a silica. The invention also provides compositions of manufacture and detergents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/103,276, filed May 9, 2011, currently pending; which is a divisionalof U.S. patent application Ser. No. 11/570,169, filed Nov. 2, 2007, nowabandoned; which is a National Stage Filing of International ApplicationNo.: PCT/US2005/032351, filed Sep. 9, 2005; which claims the benefit ofU.S. Provisional Application No. 60/609,125, filed Sep. 10, 2004, nowexpired; all of the above mentioned patents are hereby incorporated intheir entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

A Sequence Listing is submitted herewith as an ASCII-compliant text filenamed “2015-03-27-seqlisting.txt_(—)2919208-186001”, created Mar. 25,2015, and having a size of 52,344 bytes, as permitted under 37 C.F.R.§1.821(c). The material in the aforementioned text file is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to the fields of industrial processing of foods,feeds or vegetable oils, plant and animal products, and enzymology. Inparticular, the invention provides compositions and methods for theenzymatic treatment (“bleaching” or “de-colorizing”) ofchlorophyll-containing or chlorophyll-contaminated compositions, e.g.,algal, animal or plant preparations, foods, feeds or oils, for example,vegetable oils, including oils processed from oilseeds, such as canola(rapeseed) oil or soybean oil, or oil fruits, such as palm oil. In oneaspect, the invention provides methods using enzymes from chlorophyllcatabolism (e.g., a chlorophyllase) for the enzymatic modification of achlorophyll, e.g., in an algal, animal or plant preparation, or a food,a feed or an oil.

BACKGROUND

Vegetable oils coming from oilseeds such as canola or soybean oroilfruits such as palm contain chlorophyll. Chlorophyll is removedduring many stages of the oil production process, including seedcrushing, oil extraction, degumming, caustic treatment and bleachingsteps. In the last of these, the bleaching process residual chlorophyllis removed to achieve acceptable levels. This chlorophyll is typicallyremoved from the oil in a bleaching process step involving heating theoil and running it through an adsorbent to remove chlorophyll and othercolor-bearing compounds that impact the appearance and/or stability ofthe finished oil. This technology is also used to treat otherchlorophyll-containing oils or plant or algal preparations, such aspolyunsaturated fatty acid (PUFA) (e.g., eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA)) containing oils.

High level of chlorophyll pigments impart undesirable color and induceoxidation of oil during storage leading to a deterioration of the oil.In the edible oil processing industry, a bleaching step is employed tolower chlorophyll levels to as low as 0.1 ppm to guarantee oil qualityin terms of color and organolepticity. Typical desired finishedchlorophyll levels are between 0.02 to 0.05 ppm. This bleaching stepincreases processing cost and reduces oil yield due to entrainment inthe bleaching clay.

In plants, chlorophyllase (chlase) is the first enzyme involved inchlorophyll degradation; it catalyzes the hydrolysis of an ester bond inchlorophyll to yield chlorophyllide and phytol.

SUMMARY

The invention provides compositions and methods for enzymatic treatment(“bleaching” or “de-colorizing”) of chlorophyll-containing orchlorophyll-contaminated compositions such as plant, animals (e.g.,fish, meat preparations) or algal preparations, foods, feeds or oils,such as polyunsaturated fatty acid (PUFA)-containing or docosahexaenoicacid (DHA)-containing oils, or compositions comprising mixtures thereof.In one aspect, the “enzymatic bleaching” of the compositions and methodsof the invention comprises use of a chlorophyll modifying enzyme, e.g.,a polypeptide having chlorophyllase activity, including chlases andchlorophyll chlorophyllido-hydrolyases, and related polypeptides, or anychlorophyll catabolic enzyme. Thus, as used herein, the term “enzymaticbleaching” includes any modification of a chlorophyll molecule orequivalent, including partial or complete decolorization. In one aspect,the compositions and methods of the invention can reduce yield loss fromentrainment and fat splitting attributed to catalysis by clay/bleachconditions.

In alternative aspects of the methods and processes of the invention, achlorophyllase, which can be a novel chlorophyllase of the invention, ora known enzyme, including chlases and chlorophyllchlorophyllido-hydrolyases and related polypeptides, or a combinationthereof, or any chlorophyll catabolic enzyme, is added anytime oranywhere in the method or process, e.g., as discussed herein. Forexample, in one aspect, the chlorophyllase (which can be a novelchlorophyllase of the invention, or a known enzyme, or a combinationthereof) and/or any chlorophyll catabolic enzyme can be added into acomposition, such as a crude oil, with or without another enzyme, e.g.,a phospholipase (e.g., phospholipase C) at a mixing step or in adegumming step, in a caustic tank step, in a static mixer, in a day tankor in a retention mixer. Alternatively, in one aspect of a method orprocess of the invention the chlorophyllase (of the invention, or known)and/or any chlorophyll catabolic enzyme can be added into anycombination of these steps, or in all of these steps.

In one aspect, the invention provides methods or processes for enzymaticmodification of chlorophyll to facilitate its removal from acomposition, e.g., through an aqueous separation process, as illustratedin page 1, Appendix A, or hydrophobic separation process, or affinityseparation process, and the like.

In one aspect the invention provides methods and processes comprisingenzymatic modification (e.g., catabolism) of chlorophyll, or equivalentcompounds, in a composition (e.g., a food, feed, plant, animal, algae,etc.) further comprising removal of components of that composition(e.g., compounds not desirable in a finished product), such as residualchlorophyll (e.g., chlorophyll or equivalent compounds not modified by achlorophyllase), a pesticide, a polycyclic aromatic hydrocarbon, etc.Undesirable components, e.g., residual chlorophyll, pesticides,polycyclic aromatic hydrocarbons and the like, can be removed witheither significantly smaller amounts of bleaching clay or otheradsorbent, such as silica or equivalent compounds.

In one aspect, these components of the composition are removed using ableaching clay, e.g., in a plurality of steps using bleaching clay,where in one aspect components of the composition are removed witheither significantly smaller amounts of bleaching clay and/or at leastone other adsorbent (e.g., a silica). In one aspect, finishedchlorophyll levels are between about 0.02 ppm to 0.05 ppm. In thisexemplary process, the bleaching step can increase processing costs andreduce oil yields due to entrainment in the bleaching clay. Thecompositions and processes of the invention can reduce yield loss fromentrainment and fat splitting attributed to catalysis by clay/bleachconditions.

In an exemplary illustrated method (reaction) of the invention, achlorophyllase catalyzes the hydrolysis of chlorophyll to generatechlorophyllide, which in one aspect is aqueous extracted, and phytol,which remains in the oil phase. In another exemplary method,pheophorbide can be removed in manner similar to chlorophyllide. In oneaspect, by practicing the compositions and methods of the invention, anaqueous separation process can partially or completely eliminate theneed for adsorbants. However, in another aspect, the methods comprisepartial or complete extraction of the aqueous soluble chlorophyllide orpheophorbide using a silica-based extraction process (e.g.,adsorbent-free or reduced adsorbent silica refining). In one aspect, thechlorophyllase is immobilized onto a silica (which then adsorbs thechlorophyllide), e.g., a silica gel. In one aspect, the silica comprisesa TriSyl Silica or a SORBSIL R™ silica.

The invention provides methods, including industrial processes, forenzymatic treatment of pheophytin-containing or pheophytin-contaminatedcompositions comprising the following steps: (a) providing apheophytin-containing or pheophytin-contaminated composition; (b)providing a polypeptide having a chlorophyllase or pheophytinaseactivity (which can be a novel chlorophyllase of the invention, or aknown enzyme, or a combination thereof); and (c) reacting thecomposition of step (a) with the polypeptide of step (b) underconditions wherein the polypeptide can catalyze a pheophytin-modifyingreaction. The magnesium-less derivative of chlorophyll is calledpheophytin. Pheophytin is colored and often present in oil, especiallyif acid treatment has been used. In some applications, it is desirableto remove the pheophytin. The product of chlorophyllase treatment ofpheophytin is pheophorbide, which can be removed in a similar manner tochlorophyllide.

In one aspect, the compositions and methods of the invention arepracticed as or with industrial processes, e.g., oil bleaching orcaustic neutralization or degumming processes. In one aspect, use of thecompositions and methods of the invention facilitate reducing the amountof or eliminating the need for adsorbants in current bleachingprocessing, which typically involve heating the oil or otherchlorophyll-containing composition and running it through an adsorbentto remove chlorophyll and other color-bearing compounds that impact theappearance and/or stability of the finished oil. Thus, in practicingthis aspect of the invention, by partially or completely eliminate theneed for adsorbants, processing costs can be decreased, e.g., adsorbents(e.g., clay) costs, disposal costs, water costs, energy costs, steamcosts can be decreased. Other benefits in practicing various aspects ofthe invention include yield improvements, e.g., reduced entrained oilsin adsorbent substrates, increased end product value, includingretention of valuable micronutrients such as beta carotene, processefficiencies, including reduced processing steps, capital savings and anenvironmental benefit, e.g., reducing or eliminating land-filling ofbleaching adsorbents.

In one aspect, in practicing the compositions and methods of theinvention, the chlorophyll-modifying polypeptides (which can be a novelchlorophyllase of the invention, or a known enzyme, or a combinationthereof) can be employed at any point in a degumming (e.g., enzymaticdegumming) process. For example, the chlorophyll-modifying polypeptidescan be added before or after any step in a process, or before or afterany combination of steps, or before or after all of the steps, in aprocess, e.g., prior to, during or following mechanical and/or chemicalextraction, and/or degumming and/or caustic neutralization and/orbleaching and the like.

In alternative aspects of any of the methods of the invention, at leastone step is performed in a reaction vessel, e.g., an oil degummingapparatus. In alternative aspects of any of the methods of theinvention, at least one step is performed in a cell extract. Inalternative aspects of any of the methods of the invention, at least onestep is performed in a whole cell. The cell can be of any source, e.g.,a plant cell, a bacterial cell, a fungal cell, an animal cell (e.g., amammalian cell, a fish cell) or a yeast cell.

The invention provides methods for enzymatic treatment ofchlorophyll-containing or chlorophyll-contaminated compositionscomprising the following steps: (a) providing a chlorophyll-containingor chlorophyll-contaminated composition (which can be a novelchlorophyllase of the invention, or a known enzyme, or a combinationthereof); (b) providing a polypeptide having a chlorophyllase activity;and (c) reacting the composition of step (a) with the polypeptide ofstep (b) under conditions wherein the polypeptide can catalyze achlorophyll-modifying reaction.

The invention provides industrial processes for enzymatic treatment(“bleaching”) of chlorophyll-containing or chlorophyll-contaminatedcompositions comprising the following steps: (a) providing achlorophyll-containing or chlorophyll-contaminated composition (whichcan be a novel chlorophyllase of the invention, or a known enzyme, or acombination thereof); (b) providing a polypeptide having chlorophyllaseactivity; and (c) reacting the composition of step (a) with thepolypeptide of step (b) under conditions wherein the polypeptide cancatalyze a chlorophyll-modifying reaction.

The invention provides degumming processes comprising a step forenzymatic bleaching of chlorophyll-containing orchlorophyll-contaminated compositions comprising the following steps:(a) providing a chlorophyll-containing or chlorophyll-contaminatedcomposition (which can be a novel chlorophyllase of the invention, or aknown enzyme, or a combination thereof); (b) providing a polypeptidehaving chlorophyllase activity; and (c) reacting the composition of step(a) with the polypeptide of step (b) under conditions wherein thepolypeptide can catalyze a chlorophyll-modifying reaction.

There is a second ester on chlorophylls and pheophytins—a methyl ester.The methods of the invention can further comprise hydrolysis of thismethyl ester by an esterase. This can increase the tendency of thereaction derivative (now a diacid) to partition into an aqueous layer.

In an exemplary method, a phospholipase, e.g., a phospholipase C, oranother hydrolase (e.g., a cellulase, a hemicellulase, an esterase, aprotease and/or a phosphatase) is used, e.g., to improve oil extractionand oil degumming.

In alternative aspects, the methods and processes of the invention canfurther comprise hydrolysis of methyl ester on a chlorophyll or apheophytin by an esterase (which can be a novel enzyme of the invention,or a known enzyme, or a combination thereof). In alternative aspects,the methods of the invention can further comprise removal of themodified chlorophyll in an aqueous extraction. The methods can furthercomprise modifying pH (e.g., increasing pH) to promote aqueousseparation of chlorophyllide. The enzymes used in the methods, e.g., achlorophyllase, can be added during this increased pH, or “caustic”phase in the separation process. The methods can further comprise acaustic neutralization step. The methods can further comprise anadsorbent-free or reduced adsorbent silica refining step to remove achlorophilide generated by the enzymatic degradation of the chlorophyll.The methods can further comprise use of a hydrolase, e.g., aphospholipase C.

In one aspect of the methods and processes, the polypeptide is anesterase (e.g., an enzyme of the invention), e.g., a chlorophyllase, orhas chlorophyllase-like activity, or has chlorophyll catabolic activity.In one aspect of the methods, the polypeptide is immobilized. Thepolypeptide can be immobilized on an inorganic support or organicsupport. The inorganic support can comprise alumina, celite,Dowex-1-chloride, glass beads or silica gel or equivalent. Thepolypeptide can be immobilized on an alginate hydrogel or alginate beador equivalent. In one aspect of the methods, the polypeptide furthercomprises a liposome, a hydrogel or a gel.

In one aspect of the methods, the polypeptide is at least one step isperformed in a reaction vessel, e.g., a vessel comprising agravitational gum separation device or a holding tank or the like. Inone aspect of the methods, at least one step is performed in a cellextract, or a whole cell. The cell can be a plant cell, a bacterialcell, a fungal cell, a yeast cell, a mammalian cell, an insect cell andthe like.

In one aspect of the methods, the chlorophyll-containing orchlorophyll-contaminated composition comprises a plant material, plantoil or plant extract. The plant material, plant oil or plant extract cancomprise a vegetable oil or a seed oil. The vegetable oil can comprise apalm oil or a canola oil. Alternatively, the plant material, plant oilor plant extract can comprise an algal preparation. In one aspect of themethods, the chlorophyll-containing or chlorophyll-contaminatedcompositions comprise a non-wood or wood product. In one aspect of themethods, the chlorophyll-containing or chlorophyll-contaminatedcompositions comprise a fabric or cloth. In one aspect of the methods,the chlorophyll-containing or chlorophyll-contaminated compositionscomprise a pharmaceutical formulation, a food, an oil, a feed, or adietary supplement.

The compositions and methods of the invention can be used to treat crudeor refined oils, e.g., oils derived from plant (e.g., vegetable), algae,animal or fish, or synthetic, sources. The compositions and methods ofthe invention can be used to treat crude or refined oils at higher oilconcentrations, or, in one aspect, used to treat unrefined andnon-diluted crude oils.

In one aspect the methods further comprise removal of a chlorophilidegenerated by enzymatic degradation of a chlorophyll by adsorbing onto asilica gel or equivalent. The chlorophyll-containing orchlorophyll-contaminated compositions can comprise a textile, cloth,thread or fabric or related composition, a wood or paper product orby-product, such as a wood pulp, a paper pulp, a Kraft pulp, or, anon-wood paper product or by-product, such as a rice paper.

The invention provides products of manufacture comprising a degummingsystem for the enzymatic treatment of chlorophyll-containing orchlorophyll-contaminated compositions comprising: (a) a vegetable oilrefining apparatus; and (b) a polypeptide having chlorophyllase activity(e.g., an enzyme of the invention), wherein the activity of thepolypeptide comprises catalysis of a chlorophyll-modifying reaction, andthe vegetable oil refining apparatus can react a chlorophyll-containingor chlorophyll-contaminated composition with the polypeptide to underconditions wherein the polypeptide can catalyze a chlorophyll-modifyingreaction. In one aspect of the product of manufacture, the vegetable oilrefining apparatus comprises an oil leaving expellor, a holding tank ora gravitational gum separation device. The chlorophyll-modifyingreactions can comprise generation of chlorophyllide and phytol.

The invention provides detergents comprising an enzymatic treatment ofchlorophyll-containing or chlorophyll-contaminated fabrics comprising:(a) a detergent composition; and (b) a polypeptide having chlorophyllaseactivity (e.g., an enzyme of the invention), wherein the activitycomprises catalysis of a chlorophyll-modifying reaction. In one aspect,the chlorophyll-modifying reaction comprises generation ofchlorophyllide and phytol.

The invention provides methods for enzymatically treating achlorophyll-containing or chlorophyll-contaminated fabrics comprising:(a) providing a detergent composition comprising a polypeptide havingchlorophyllase activity (e.g., an enzyme of the invention), wherein theactivity comprises catalysis of a chlorophyll-modifying reaction; and,(b) contacting the detergent composition with the chlorophyll-containingor chlorophyll-contaminated fabric under conditions wherein thepolypeptide can catalyze a chlorophyll-modifying reaction. In oneaspect, the chlorophyll-modifying reaction comprises generation ofchlorophyllide and phytol.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19, over a region of at leastabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100,2200, 2250, 2300, 2350, 2400, 2450, 2500, or more residues, sequencessubstantially identical thereto, and the sequences complementarythereto, and encodes at least one polypeptide having an enzymaticactivity as described herein, e.g., an esterase enzyme activity.

In alternative aspects, the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.In one aspect, the sequence comparison algorithm is a BLAST algorithm,e.g., a BLAST version 2.2.2 algorithm where a filtering setting is setto blastall -p blastp -d “nr pataa”-F F, and all other options are setto default.

Exemplary nucleic acids of the invention also include isolated,synthetic or recombinant nucleic acids encoding a polypeptide of theinvention, e.g., a polypeptide having a sequence as set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20, andsubsequences thereof and variants thereof.

In alternative aspects, the polypeptide has an esterase enzyme activity,including chlorophyllase (a chlase) activity, or, enzyme activitycomprising enzymatic modification of a chlorophyll molecule, e.g.,wherein the enzymatic modification comprises catabolism of thechlorophyll molecule. In one aspect, the esterase activity comprises achlorophyll chlorophyllido-hydrolyase activity.

In one aspect, the isolated, synthetic or recombinant nucleic acid ofthe invention encodes a polypeptide having an enzyme activity that isthermostable. The polypeptide can retain enzyme activity underconditions comprising a temperature range of between about 37° C. toabout 95° C.; between about 55° C. to about 85° C., between about 70° C.to about 95° C., or, between about 90° C. to about 95° C.

In another aspect, an isolated, synthetic or recombinant nucleic acid ofthe invention encodes a polypeptide having enzyme that isthermotolerant. The polypeptide can retain enzyme activity afterexposure to a temperature in the range from greater than 37° C. to about95° C. or anywhere in the range from greater than 55° C. to about 85° C.The polypeptide can retain enzyme activity after exposure to atemperature in the range between about 1° C. to about 5° C., betweenabout 5° C. to about 15° C., between about 15° C. to about 25° C.,between about 25° C. to about 37° C., between about 37° C. to about 95°C., between about 55° C. to about 85° C., between about 70° C. to about75° C., or between about 90° C. to about 95° C., or more. In one aspect,the polypeptide retains enzyme activity after exposure to a temperaturein the range from greater than 90° C. to about 95° C. at pH 4.5.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a sequence that hybridizes under stringent conditions to anucleic acid comprising a sequence of the invention, e.g., a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ IDNO:19, or fragments or subsequences thereof (or complements thereof). Inone aspect, the nucleic acid of the invention encodes a polypeptidehaving an esterase enzyme activity, including chlorophyllase (a chlase)activity, or, enzyme activity comprising enzymatic modification of achlorophyll molecule, e.g., wherein the enzymatic modification comprisescatabolism of the chlorophyll molecule. In one aspect, the esteraseactivity comprises a chlorophyll chlorophyllido-hydrolyase activity. Thenucleic acid can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more residuesin length or the full length of the gene or transcript. In one aspect,the stringent conditions include a wash step comprising a wash in0.2×SSC at a temperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an enzyme activity as describedherein (e.g., esterase enzyme activity, including chlorophyllase (achlase) activity), wherein the probe comprises at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000 or more, consecutive bases of a sequence comprisinga sequence of the invention, or fragments or subsequences thereof,wherein the probe identifies the nucleic acid by binding orhybridization. The probe can comprise an oligonucleotide comprising atleast about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, orabout 60 to 100 consecutive bases of a sequence comprising a sequence ofthe invention, or fragments or subsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having at least one enzyme activity asdescribed herein (e.g., esterase enzyme activity, includingchlorophyllase (a chlase) activity), wherein the probe comprises anucleic acid comprising a sequence at least about 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to a nucleic acid of the invention, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence of the invention, or asubsequence thereof.

The invention provides an amplification primer pair for amplifying anucleic acid encoding a polypeptide having at least one enzyme activityas described herein (e.g., esterase enzyme activity, includingchlorophyllase (a chlase) activity), wherein the primer pair is capableof amplifying a nucleic acid comprising a sequence of the invention, orfragments or subsequences thereof. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence, orabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of the complementary strand ofthe first member.

The invention provides nucleic acids encoding proteins (e.g., enzymes),including the polypeptides of the invention, generated by amplification,e.g., polymerase chain reaction (PCR), using an amplification primerpair of the invention. The invention provides nucleic acids encodingpolypeptides having at least one enzyme activity as described herein(e.g., esterase enzyme activity, including chlorophyllase (a chlase)activity) using an amplification primer pair of the invention. Theinvention provides methods of making and/or identifying enzymes byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having enzyme activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a cereal, a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a cereal plant, a corn plant, apotato plant, a tomato plant, a wheat plant, an oilseed plant, arapeseed plant, a soybean plant, a rice plant, a barley plant or atobacco plant. The invention provides transgenic seeds comprising anucleic acid of the invention or an expression cassette (e.g., a vector)of the invention. The transgenic seed can be a cereal plant, a cornseed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palmkernel, a sunflower seed, a sesame seed, a peanut or a tobacco plantseed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a enzyme message (of an enzymeof the invention) in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. In one aspect, theantisense oligonucleotide is between about 10 to 50, about 20 to 60,about 30 to 70, about 40 to 80, or about 60 to 100 bases in length.

The invention provides methods of inhibiting the translation of anenzyme message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesdouble-stranded inhibitory RNA (RNAi) molecules comprising a subsequenceof a sequence of the invention. In one aspect, the RNAi is about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.The invention provides methods of inhibiting the expression of apolypeptide (e.g., an enzyme of the invention) in a cell comprisingadministering to the cell or expressing in the cell a double-strandedinhibitory RNA (iRNA), wherein the RNA comprises a subsequence of asequence of the invention.

The invention provides an isolated, synthetic or recombinant polypeptidecomprising an amino acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary polypeptide or peptide of the invention over a region ofat least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350 or more residues, or overthe full length of the polypeptide. In one aspect, the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection. Exemplary polypeptide or peptidesequences of the invention include SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18 or SEQ ID NO:20, and subsequences thereof andvariants thereof. Exemplary polypeptides also include fragments of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more residues inlength, or over the full length of an enzyme. Exemplary polypeptide orpeptide sequences of the invention include sequence encoded by a nucleicacid of the invention. Exemplary polypeptide or peptide sequences of theinvention include polypeptides or peptides specifically bound by anantibody of the invention. The peptide can be, e.g., an immunogenicfragment, a motif (e.g., a binding site), a signal sequence, a preprosequence, a catalytic domains (CDs) or an active site.

In one aspect, a polypeptide of the invention has an esterase activity,such as a chlorophyllase (a chlase) activity, or, has an enzyme activitycomprising enzymatic modification of a chlorophyll molecule, e.g.,wherein the enzymatic modification comprises catabolism of thechlorophyll molecule. In one aspect, the esterase activity comprises achlorophyll chlorophyllido-hydrolyase activity.

Another aspect of the invention provides an isolated, synthetic orrecombinant polypeptide or peptide including at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or moreconsecutive bases of a polypeptide or peptide sequence of the invention,sequences substantially identical thereto, and the sequencescomplementary thereto. The peptide can be, e.g., an immunogenicfragment, a motif (e.g., a binding site), a signal sequence, a preprosequence or a catalytic domains (CDs) or active site.

The invention provides biosynthetic systems comprising nucleic acidsand/or plasmids of the invention in a cell, e.g., a yeast cell, a plantcell, a fungal cell, or a microbial (e.g., bacterial) cell. In oneaspect, the biosynthetic systems of the invention comprise codingsequences for all the enzymes necessary, or a subset thereof, forcatabolism of a chlorophyll molecule. In one aspect, the codingsequences can be in a plasmid, a recombinant vector or virus and thelike.

In one aspect, the enzyme activity of a polypeptide of the invention isthermostable. The polypeptide of the invention can retain activity underconditions comprising a temperature range of between about 1° C. toabout 5° C., between about 5° C. to about 15° C., between about 15° C.to about 25° C., between about 25° C. to about 37° C., between about 37°C. to about 95° C., between about 55° C. to about 85° C., between about70° C. to about 75° C., or between about 90° C. to about 95° C., ormore. In another aspect, the enzyme activity of a polypeptide of theinvention is thermotolerant. The polypeptide can retain activity afterexposure to a temperature in the range from greater than 37° C. to about95° C., or in the range from greater than 55° C. to about 85° C. In oneaspect, the polypeptide can retain activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

In one aspect, the isolated, synthetic or recombinant polypeptide cancomprise the polypeptide of the invention that lacks a signal sequence.In one aspect, the isolated, synthetic or recombinant polypeptide cancomprise the polypeptide of the invention comprising a heterologoussignal sequence.

In one aspect, the invention provides chimeric proteins comprising afirst domain comprising a signal sequence of the invention and at leasta second domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The chimeric enzyme can comprise all or asubsequence of at least one polypeptide having an activity as describedherein (e.g., esterase enzyme activity, including chlorophyllase (achlase) activity).

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) of the invention and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro sequence and/or catalytic domain (CD). Inone aspect, the heterologous polypeptide or peptide is not a polypeptidehaving activity comprising esterase enzyme activity or chlorophyllcatabolism activity. The heterologous polypeptide or peptide can beamino terminal to, carboxy terminal to or on both ends of the signalpeptide (SP), prepro sequence and/or catalytic domain (CD).

The invention provides isolated, synthetic or recombinant nucleic acidsencoding a chimeric polypeptide, wherein the chimeric polypeptidecomprises at least a first domain comprising signal peptide (SP), aprepro domain and/or a catalytic domain (CD) of the invention and atleast a second domain comprising a heterologous polypeptide or peptide,wherein the heterologous polypeptide or peptide is not naturallyassociated with the signal peptide (SP), prepro domain and/or catalyticdomain (CD).

The invention provides isolated, synthetic or recombinant signalsequences (e.g., signal peptides) consisting of or comprising a sequenceas set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43 or1 to 44, of a polypeptide of the invention, e.g., SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20. The inventionprovides isolated, synthetic or recombinant signal sequences (e.g.,signal peptides) consisting of or comprising a sequence as set forth inTable 1, below.

In one aspect, an enzyme of the invention has a specific activity atabout 37° C. in the range from about 1 to about 1200 units per milligramof protein, or, about 100 to about 1000 units per milligram of protein.In another aspect, an enzyme of the invention has a specific activityfrom about 100 to about 1000 units per milligram of protein, or, fromabout 500 to about 750 units per milligram of protein. Alternatively, anenzyme of the invention has a specific activity at 37° C. in the rangefrom about 1 to about 750 units per milligram of protein, or, from about500 to about 1200 units per milligram of protein. In one aspect, anenzyme of the invention has a specific activity at 37° C. in the rangefrom about 1 to about 500 units per milligram of protein, or, from about750 to about 1000 units per milligram of protein. In another aspect, anenzyme of the invention has a specific activity at 37° C. in the rangefrom about 1 to about 250 units per milligram of protein. Alternatively,an enzyme of the invention has comprises a specific activity at 37° C.in the range from about 1 to about 100 units per milligram of protein.In another aspect, the thermotolerance comprises retention of at leasthalf of the specific activity of the enzyme at 37° C. after being heatedto the elevated temperature. Alternatively, the thermotolerance cancomprise retention of specific activity at 37° C. in the range fromabout 1 to about 1200 units per milligram of protein, or, from about 500to about 1000 units per milligram of protein, after being heated to theelevated temperature. In another aspect, the thermotolerance cancomprise retention of specific activity at 37° C. in the range fromabout 1 to about 500 units per milligram of protein after being heatedto the elevated temperature.

The invention provides an isolated, synthetic or recombinant polypeptideof the invention, wherein the polypeptide comprises at least oneglycosylation site. In one aspect, glycosylation can be an N-linkedglycosylation. In one aspect, the polypeptide can be glycosylated afterbeing expressed in a P. pastoris or a S. pombe.

In one aspect, a polypeptide of the invention can retain enzyme activityunder conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 orpH 4 or more acidic. In another aspect, a polypeptide of the inventionretains activity under conditions comprising about pH 7, pH 7.5 pH 8.0,pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more basic. In oneaspect, a polypeptide of the invention retains activity under conditionscomprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or moreacidic. In another aspect, a polypeptide of the invention retainsactivity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5,pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more basic.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. In one aspect, the secondmember of the heterodimer is not a polypeptide of the invention butrather is a different enzyme or another protein. In one aspect, thesecond domain can be a polypeptide and the heterodimer can be a fusionprotein. In one aspect, the second domain can be an epitope or a tag. Inone aspect, the invention provides homodimers comprising a polypeptideof the invention.

The invention provides immobilized polypeptides of the invention, apolypeptide encoded by a nucleic acid of the invention, or a polypeptidecomprising a polypeptide of the invention and a second domain. In oneaspect, the polypeptide can be immobilized on a cell, a metal, a resin,a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, abead, a gel, a plate, an array or a capillary tube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a polypeptide of the invention or to apolypeptide encoded by a nucleic acid of the invention. The antibody canbe a monoclonal or a polyclonal antibody. The invention provideshybridomas comprising an antibody of the invention, e.g., an antibodythat specifically binds to a polypeptide of the invention or to apolypeptide encoded by a nucleic acid of the invention.

The invention provides method of isolating or identifying a polypeptideinvolved in chlorophyll catabolism or having an esterase (e.g.,chlorophyllase) activity, wherein the method comprises the steps of: (a)providing an antibody of the invention; (b) providing a samplecomprising polypeptides; and (c) contacting the sample of step (b) withthe antibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifyingthe polypeptide.

The invention provides methods of making an antibody that specificallybinds to a polypeptide of the invention (e.g., an enzyme or anotherantibody of the invention) comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate a humoralimmune response, thereby generating an antibody response. The inventionprovides methods of making a humoral or cellular immune responsecomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate an immune response.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide involved inchlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity, comprising the following steps: (a) providing a polypeptide ofthe invention; or a polypeptide encoded by a nucleic acid of theinvention; (b) providing an appropriate substrate (e.g., substrate ofthe polypeptide; and (c) contacting the polypeptide or a fragment orvariant thereof of step (a) with the substrate of step (b) and detectinga decrease in the amount of substrate or an increase in the amount of areaction product, wherein a decrease in the amount of the substrate oran increase in the amount of the reaction product detects a polypeptideinvolved in chlorophyll catabolism or having an esterase (e.g.,chlorophyllase) activity.

The invention provides methods for identifying a substrate of apolypeptide involved in a chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity, wherein the method comprises thefollowing steps: (a) providing a polypeptide of the invention; or apolypeptide encoded by a nucleic acid of the invention; (b) providing atest substrate; and (c) contacting the polypeptide of step (a) with thetest substrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofa reaction product identifies the test substrate a substrate of apolypeptide involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of apolypeptide involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity, comprising the following steps: (a)providing a polypeptide of the invention or a polypeptide encoded by anucleic acid of the invention; (b) providing a test compound; (c)contacting the polypeptide of step (a) with the test compound of step(b) and measuring an activity of the polypeptide wherein a change inactivity measured in the presence of the test compound compared to theactivity in the absence of the test compound provides a determinationthat the test compound modulates polypeptide activity. In one aspect,polypeptide activity can be measured by providing an appropriatesubstrate (e.g., substrate of the polypeptide involved in a chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity) anddetecting a decrease in the amount of the substrate or an increase inthe amount of a reaction product, or, an increase in the amount of thesubstrate or a decrease in the amount of a reaction product. A decreasein the amount of the substrate or an increase in the amount of thereaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of activity. An increase in the amount ofthe substrate or a decrease in the amount of the reaction product withthe test compound as compared to the amount of substrate or reactionproduct without the test compound identifies the test compound as aninhibitor of activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having enzymatic activity involved inchlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity from an environmental sample comprising the steps of: (a)providing an amplification primer sequence pair for amplifying a nucleicacid encoding a polypeptide involved in chlorophyll catabolism or havingan esterase (e.g., chlorophyllase) activity, wherein the primer pair iscapable of amplifying a nucleic acid of the invention; (b) isolating anucleic acid from the environmental sample or treating the environmentalsample such that nucleic acid in the sample is accessible forhybridization to the amplification primer pair; and, (c) combining thenucleic acid of step (b) with the amplification primer pair of step (a)and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide involvedin chlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity from an environmental sample. In one aspect, one or each memberof the amplification primer pair can comprise an oligonucleotidecomprising at least about 10 to 50 or more consecutive bases of asequence of the invention. In one aspect, the amplification primer pairis an amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide involved in chlorophyll catabolism or havingan esterase (e.g., chlorophyllase) activity from an environmental samplecomprising the steps of: (a) providing a polynucleotide probe comprisinga nucleic acid of the invention or a subsequence thereof; (b) isolatinga nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to a polynucleotide probe of step (a); (c) combiningthe isolated, synthetic nucleic acid or the treated environmental sampleof step (b) with the polynucleotide probe of step (a); and (d) isolatinga nucleic acid that specifically hybridizes with the polynucleotideprobe of step (a), thereby isolating or recovering a nucleic acidencoding a polypeptide involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity from an environmental sample.The environmental sample can comprise a water sample, a liquid sample, asoil sample, an air sample or a biological sample. In one aspect, thebiological sample can be derived from a bacterial cell, a protozoancell, an insect cell, a yeast cell, a plant cell, a fungal cell or amammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity comprising the steps of: (a)providing a template nucleic acid comprising a nucleic acid of theinvention; and (b) modifying, deleting or adding one or more nucleotidesin the template sequence, or a combination thereof, to generate avariant of the template nucleic acid. In one aspect, the method canfurther comprise expressing the variant nucleic acid to generate avariant polypeptide involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity. The modifications, additionsor deletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until apolypeptide involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity having an altered or different activityor an altered or different stability from that of a polypeptide encodedby the template nucleic acid is produced. In one aspect, the variantpolypeptide is thermotolerant, and retains some activity after beingexposed to an elevated temperature. In another aspect, the variantpolypeptide has increased glycosylation as compared to the polypeptideencoded by a template nucleic acid. Alternatively, the variantpolypeptide has activity under a high (or higher) temperature, whereinthe enzyme encoded by the template nucleic acid is not active under thehigh temperature. In one aspect, the method can be iteratively repeateduntil an enzyme coding sequence having an altered codon usage from thatof the template nucleic acid is produced. In another aspect, the methodcan be iteratively repeated until an enzyme-encoding gene having higheror lower level of message expression or stability from that of thetemplate nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity to increase its expression in ahost cell, the method comprising the following steps: (a) providing anucleic acid of the invention encoding a polypeptide involved inchlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity; and, (b) identifying a non-preferred or a less preferred codonin the nucleic acid of step (a) and replacing it with a preferred orneutrally used codon encoding the same amino acid as the replaced codon,wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity; the method comprising thefollowing steps: (a) providing a nucleic acid of the invention; and, (b)identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding thepolypeptide.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having enzymatic activity involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity toincrease its expression in a host cell, the method comprising thefollowing steps: (a) providing a nucleic acid of the invention encodinga polypeptide having enzymatic activity involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity; and,(b) identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having enzymatic activity involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity todecrease its expression in a host cell, the method comprising thefollowing steps: (a) providing a nucleic acid of the invention; and (b)identifying at least one preferred codon in the nucleic acid of step (a)and replacing it with a non-preferred or less preferred codon encodingthe same amino acid as the replaced codon, wherein a preferred codon isa codon over-represented in coding sequences in genes in a host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified polypeptide active sites (catalyticdomains (CDs)) or substrate binding sites of polypeptides havingenzymatic activity involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity, wherein the modified activesites or substrate binding sites are derived from a first nucleic acidcomprising a sequence encoding a first active site or a first substratebinding site the method comprising the following steps: (a) providing afirst nucleic acid encoding a first active site or first substratebinding site, wherein the first nucleic acid sequence comprises asequence that hybridizes under stringent conditions to a nucleic acid ofthe invention, and the nucleic acid encodes an active site or asubstrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified active sites or substrate binding sites ofpolypeptides having enzymatic activity involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity. In oneaspect, the method comprises mutagenizing the first nucleic acid of step(a) by a method comprising an optimized directed evolution system, GeneSite-Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, syntheticligation reassembly (SLR) and a combination thereof. In another aspect,the method comprises mutagenizing the first nucleic acid of step (a) orvariants by a method comprising recombination, recursive sequencerecombination, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesis,chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises a polypeptide of the invention or is encoded by anucleic acid of the invention; (b) providing a substrate for at leastone of the enzymes of step (a); and (c) reacting the substrate of step(b) with the enzymes under conditions that facilitate a plurality ofbiocatalytic reactions to generate a small molecule by a series ofbiocatalytic reactions. The invention provides methods for modifying asmall molecule comprising the following steps: (a) providing an enzyme,wherein the enzyme comprises a polypeptide of the invention, or, apolypeptide encoded by a nucleic acid of the invention, or a subsequencethereof; (b) providing a small molecule; and (c) reacting the enzyme ofstep (a) with the small molecule of step (b) under conditions thatfacilitate an enzymatic reaction catalyzed by the polypeptide of theinvention, or, a polypeptide encoded by a nucleic acid of the invention,thereby modifying a small molecule. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by an enzyme of theinvention. In one aspect, the method can comprise a plurality ofadditional enzymes under conditions that facilitate a plurality ofbiocatalytic reactions by the enzymes to form a library of modifiedsmall molecules produced by the plurality of enzymatic reactions. Inanother aspect, the method can further comprise the step of testing thelibrary to determine if a particular modified small molecule thatexhibits a desired activity is present within the library. The step oftesting the library can further comprise the steps of systematicallyeliminating all but one of the biocatalytic reactions used to produce aportion of the plurality of the modified small molecules within thelibrary by testing the portion of the modified small molecule for thepresence or absence of the particular modified small molecule with adesired activity, and identifying at least one specific biocatalyticreaction that produces the particular modified small molecule of desiredactivity.

The invention provides methods for determining a functional fragment ofa polypeptide of the invention, or, a polypeptide encoded by a nucleicacid of the invention, comprising the steps of: (a) providing apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention, or a subsequence thereof; and (b) deleting a pluralityof amino acid residues from the sequence of step (a) and testing theremaining subsequence for enzyme or binding activity, therebydetermining a functional fragment of the enzyme. In one aspect, activityis measured by providing a substrate and detecting a decrease in theamount of the substrate or an increase in the amount of a reactionproduct.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of a polypeptide of the invention, or, a polypeptideencoded by a nucleic acid of the invention, the method comprisingglycosylating a polypeptide comprising at least thirty contiguous aminoacids of a polypeptide of the invention; or a polypeptide encoded by anucleic acid sequence of the invention, thereby increasing thethermotolerance or thermostability of the polypeptide. In one aspect,the specific activity can be thermostable or thermotolerant at atemperature in the range from greater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinantpolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell. The invention provides methods of expressing aheterologous nucleic acid sequence in a plant cell comprising thefollowing steps: (a) transforming the plant cell with a heterologousnucleic acid sequence operably linked to a promoter, wherein theheterologous nucleic sequence comprises a sequence of the invention; (b)growing the plant under conditions wherein the heterologous nucleicacids sequence is expressed in the plant cell. In another aspect, anenzyme of the invention can be prepared by expression of apolynucleotide of the invention in an organism such as a bacterium, ayeast, a plant, an insect, a fungus or an animal. Exemplary organismsfor expressing polypeptides of the invention can be S. pombe, S.cerevisiae, Pichia sp., e.g., P. pastoris, E. coli, Streptomyces sp.,Bacillus sp. and Lactobacillus sp.

Another aspect of the invention is a method of making a polypeptide ofthe invention. The method includes introducing a nucleic acid encodingthe polypeptide into a host cell, wherein the nucleic acid is operablylinked to a promoter and culturing the host cell under conditions thatallow expression of the nucleic acid. Another aspect of the invention isa method of making a polypeptide or peptide of the invention. The methodincludes introducing a nucleic acid encoding the polypeptide into a hostcell, wherein the nucleic acid is operably linked to a promoter andculturing the host cell under conditions that allow expression of thenucleic acid, thereby producing the polypeptide.

Another aspect of the invention is a method of generating a variantincluding obtaining a nucleic acid having a sequence of the invention,sequences substantially identical thereto, sequences complementary to asequence of the invention, and fragments thereof, and changing one ormore nucleotides in the sequence to another nucleotide, deleting one ormore nucleotides in the sequence, or adding one or more nucleotides tothe sequence.

The invention provides biosynthetic systems for the catabolism ofchlorophyll comprising at least one enzyme of the invention. Theinvention provides biosynthetic systems for the catabolism ofchlorophyll comprising at least one nucleic acid encoding an enzymeinvolved in the catabolism of chlorophyll, wherein the nucleic acidcomprises a sequence of the invention. In one aspect, the systemcomprises a plurality of enzyme-encoding nucleic acids, wherein theenzymes are involved in the catabolism of chlorophyll. In one aspect,the plurality of enzyme-encoding nucleic acids comprises all of theenzymes in a chlorophyll catabolism pathway. In one aspect, theplurality of enzyme-encoding nucleic acids are contained in at least oneplasmid, expression cassette or expression vector.

In one aspect, the biosynthetic system of the invention is contained in(comprises) a cell. The cell can be a bacterial cell, a mammalian cell,a fungal cell, a yeast cell, an insect cell or a plant cell. The yeastcell can be a Pichia sp. or a Saccharomyces sp., such as a Pichiapastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein, including Appendix A, are hereby expresslyincorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of chlorophyll (FIG. 1A), phytol(FIG. 1B) and chlorophyllide (FIG. 1C).

FIG. 2 and FIG. 3 illustrate data showing the results of an esterase(chlorophyllase activity) activity assay using exemplary enzymes of theinvention, as described in detail in Example 1, below.

FIG. 4 is a block diagram of an exemplary computer system of theinvention, as described in detail, below.

FIG. 5 is a flow diagram illustrating one aspect of a process of theinvention for comparing a new nucleotide or protein sequence with adatabase of sequences in order to determine the homology levels betweenthe new sequence and the sequences in the database, as described indetail, below.

FIG. 6 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous, asdescribed in detail, below.

FIG. 7 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence, asdescribed in detail, below.

FIG. 8 illustrates the reaction of an exemplary esterase of theinvention in chlorophyll degradation, as described in detail, below.

FIG. 9 illustrates and compares traditional versus an exemplaryenzymatic decoloring (bleaching) reaction of the invention, as describedin detail, below.

FIG. 10 illustrates an exemplary enzymatic decoloring (bleaching)reaction of the invention, as described in detail, below.

FIG. 11 illustrates an exemplary enzymatic decoloring (bleaching)process of the invention that combines degumming, enzymatic bleaching(“decoloring”) and caustic neutralization steps, as described in detail,below.

FIG. 12 illustrates an exemplary enzymatic decoloring (bleaching)process of the invention as described in detail, below.

FIG. 13 illustrates an exemplary oilseed refining scheme comprisingextraction, refining and modification of an oilseed using an esterase ofthe invention, as described in detail, below.

FIG. 14 illustrates an exemplary industrial process of the invention—abiodegumming process, comprising use of at least one polypeptide of theinvention, as described in detail, below.

FIG. 15 illustrates another exemplary industrial process of theinvention comprising use of at least one polypeptide of the invention,as described in detail, below.

FIG. 16 illustrates another exemplary industrial process of theinvention comprising use of at least one polypeptide of the inventionhaving chlorophyllase enzyme activity.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The present invention provides novel compositions and processes for theenzymatic treatment (“bleaching” or “de-colorizing”) ofchlorophyll-containing or chlorophyll-contaminated compositions, e.g.,plant, animal or algal preparations, foods, feeds or oils. In oneaspect, the treatment (or, “enzymatic bleaching” or “de-colorizing”) ofchlorophyll used in the compositions and methods of the inventioncomprises use of a chlorophyllase enzyme, or other enzyme involved inchlorophyll catabolism, to modify chlorophyll, e.g., to facilitateremoval of the color-bearing porphyrin ring by, e.g., aqueousextraction. Chlorophyllase catalyzes the hydrolysis of chlorophyll togenerate chlorophyllide, which can be aqueous extracted, and phytol,which remains in the oil phase.

For example, in one aspect, the invention provides compositions andprocesses for the enzymatic processing (e.g., hydrolysis) of chlorophyllin a feed, food or oil, e.g., a vegetable oils, including oils processedfrom oilseeds, such as canola (rapeseed) oil or soybean oil, or oilfruits, such as palm oil. In one aspect, the invention providesenzymatic bleaching methods using a chlorophyllase enzyme for theenzymatic hydrolysis of a chlorophyll or any color-bearing porphyrinring in an animal or a plant oil, e.g., vegetable oils.

The invention includes methods for enzymatically treating (e.g.,“bleaching”) chlorophyll-containing foods or oils via in vitro or invivo techniques, e.g., whole cells protocols, such as fermentation orother biocatalytic processes.

Generating and Manipulating Nucleic Acids

The invention provides isolated, recombinant and synthetic nucleic acids(e.g., an exemplary nucleic acid of the invention, including SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19), and sequenceshaving a sequence identity to an exemplary nucleic acid; nucleic acidsencoding polypeptides of the invention, e.g., the exemplary amino acidsequences as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18 or SEQ ID NO:20). The invention also provides expression cassettessuch as expression vectors, comprising nucleic acids of the invention,which include polynucleotides which encode the polypeptides of theinvention. The invention also includes methods for discovering newpolypeptide sequences using the nucleic acids of the invention. Theinvention also includes methods for inhibiting the expression of genes,transcripts and polypeptides using the nucleic acids of the invention.Also provided are methods for modifying the nucleic acids of theinvention by, e.g., synthetic ligation reassembly, optimized directedevolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like.

In practicing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of the invention, or a fragment comprising at least 10,15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500consecutive bases of a nucleic acid of the invention. The isolated,nucleic acids may comprise DNA, including cDNA, genomic DNA andsynthetic DNA. The DNA may be double-stranded or single-stranded and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. Alternatively, the isolated nucleic acids may comprise RNA.

The isolated nucleic acids of the invention may be used to prepare oneof the polypeptides of the invention, or fragments comprising at least5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive aminoacids of one of the polypeptides of the invention.

Accordingly, another aspect of the invention is an isolated nucleic acidwhich encodes one of the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of the invention. Thecoding sequences of these nucleic acids may be identical to one of thecoding sequences of one of the nucleic acids of the invention or may bedifferent coding sequences which encode one of the of the inventionhaving at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of the invention, asa result of the redundancy or degeneracy of the genetic code. Thegenetic code is well known to those of skill in the art and can beobtained, for example, on page 214 of B. Lewin, Genes VI, OxfordUniversity Press, 1997.

The isolated nucleic acid which encodes one of the polypeptides of theinvention, but is not limited to: only the coding sequence of a nucleicacid of the invention and additional coding sequences, such as leadersequences or proprotein sequences and non-coding sequences, such asintrons or non-coding sequences 5′ and/or 3′ of the coding sequence.Thus, as used herein, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only the coding sequence forthe polypeptide as well as a polynucleotide which includes additionalcoding and/or non-coding sequence.

Alternatively, the nucleic acid sequences of the invention, may bemutagenized using conventional techniques, such as site directedmutagenesis, or other techniques familiar to those skilled in the art,to introduce silent changes into the polynucleotides o of the invention.As used herein, “silent changes” include, for example, changes which donot alter the amino acid sequence encoded by the polynucleotide. Suchchanges may be desirable in order to increase the level of thepolypeptide produced by host cells containing a vector encoding thepolypeptide by introducing codons or codon pairs which occur frequentlyin the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of the invention. Suchnucleotide changes may be introduced using techniques such as sitedirected mutagenesis, random chemical mutagenesis, exonuclease IIIdeletion and other recombinant DNA techniques. Alternatively, suchnucleotide changes may be naturally occurring allelic variants which areisolated by identifying nucleic acids which specifically hybridize toprobes comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive bases of one of the sequences of theinvention (or the sequences complementary thereto) under conditions ofhigh, moderate, or low stringency as provided herein.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids which have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone aspect, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (Seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In alternativeaspects, nucleic acids of the invention are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow), as set forth herein.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDSand 200 n/ml sheared and denatured salmon sperm DNA. Hybridization couldoccur under reduced stringency conditions as described above, but in 35%formamide at a reduced temperature of 35° C. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of an enzyme of the invention. Variants can be produced by anynumber of means included methods such as, for example, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM and any combination thereof.

The term “Saturation Mutagenesis” or “Gene Site Saturation Mutagenesis”or “GSSM” includes a method that uses degenerate oligonucleotide primersto introduce point mutations into a polynucleotide, as described indetail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense (complementary) strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin. The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide” includeseither a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide can ligate to afragment that has not been dephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons). “Operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments. Inone aspect, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system. In oneaspect, promoter transcriptional regulatory sequences that are operablylinked to a transcribed sequence are physically contiguous to thetranscribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as an enzyme of the invention) ina host compatible with such sequences. Expression cassettes include atleast a promoter operably linked with the polypeptide coding sequence;and, optionally, with other sequences, e.g., transcription terminationsignals. Additional factors necessary or helpful in effecting expressionmay also be used, e.g., enhancers. Thus, expression cassettes alsoinclude plasmids, expression vectors, recombinant viruses, any form ofrecombinant “naked DNA” vector, and the like. A “vector” comprises anucleic acid which can infect, transfect, transiently or permanentlytransduce a cell. It will be recognized that a vector can be a nakednucleic acid, or a nucleic acid complexed with protein or lipid. Thevector optionally comprises viral or bacterial nucleic acids and/orproteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). Vectors include, but are not limited to replicons(e.g., RNA replicons, bacteriophages) to which fragments of DNA may beattached and become replicated. Vectors thus include, but are notlimited to RNA, autonomous self-replicating circular or linear DNA orRNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

General Techniques

The present invention provides novel compositions and processes forenzymatically treating (e.g., “bleaching”) chlorophyll-containingcompositions such as plants, algae, foods or oils. The skilled artisanwill recognize that compounds used in the methods of the invention(e.g., catalytic, starting or intermediate compounds) can be synthesizedusing a variety of procedures and methodologies, which are welldescribed in the scientific and patent literature., e.g., OrganicSyntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons,Inc., NY; Venuti (1989) Pharm Res. 6:867-873. The invention can bepracticed in conjunction with any method or protocol known in the art,which are well described in the scientific and patent literature.

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., enzymes of the invention) generated fromthese nucleic acids can be individually isolated or cloned and testedfor a desired activity. Any recombinant expression system can be used,including bacterial, mammalian, yeast, insect or plant cell expressionsystems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

The present invention provides novel compositions and processes forenzymatically treating (e.g., “bleaching”) chlorophyll-containingcompositions such as plants, algae, foods or oils. The skilled artisanwill recognize that compounds used in the methods of the invention(e.g., catalytic, starting or intermediate compounds) can be synthesizedusing a variety of procedures and methodologies, which are welldescribed in the scientific and patent literature., e.g., OrganicSyntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons,Inc., NY; Venuti (1989) Pharm Res. 6:867-873. The invention can bepracticed in conjunction with any method or protocol known in the art,which are well described in the scientific and patent literature.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lacI promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused. Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the ladpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter.Fungal promoters include the V factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express an enzyme of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express an enzyme of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofenzyme-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the enzyme-producing nucleic acids of the invention willallow the grower to select plants with the optimal enzyme expressionand/or activity. The development of plant parts can thus controlled. Inthis way the invention provides the means to facilitate the harvestingof plants and plant parts. For example, in various embodiments, themaize In2-2 promoter, activated by benzenesulfonamide herbicidesafeners, is used (De Veylder (1997) Plant Cell Physiol. 38:568-577);application of different herbicide safeners induces distinct geneexpression patterns, including expression in the root, hydathodes, andthe shoot apical meristem. Coding sequences of the invention are alsounder the control of a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theenzymes of the invention. Expression vectors and cloning vehicles of theinvention can comprise viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as Bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lad, lacZ, T3,T7, gpt, lambda P_(R), P_(R) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors in one aspect contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli and the S. cerevisiae TRPJ gene.

In some aspects, the nucleic acid encoding one of the polypeptides ofthe invention, or fragments comprising at least about 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.Optionally, the nucleic acid can encode a fusion polypeptide in whichone of the polypeptides of the invention, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof is fused to heterologous peptides or polypeptides,such as N-terminal identification peptides which impart desiredcharacteristics, such as increased stability or simplified purification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding an enzyme of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Lactococcus lactis, Streptomyces,Bacillus subtilis, Bacillus cereus, Salmonella typhimurium or anyspecies within the genera Bacillus, Streptomyces and Staphylococcus.Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.Exemplary yeast cells include Pichia pastoris, Saccharomyces cerevisiaeor Schizosaccharomyces pombe. Exemplary animal cells include CHO, COS orBowes melanoma or any mouse or human cell line. The selection of anappropriate host is within the abilities of those skilled in the art.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S. Pat. No.5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, e.g., nucleicacids of the invention, can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides a method for overexpressing a recombinant enzymein a cell comprising expressing a vector comprising a nucleic acid ofthe invention, e.g., a nucleic acid comprising a nucleic acid sequencewith at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to an exemplary sequence of the invention over aregion of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150or more residues, or the full length of a gene or a transcript, whereinthe sequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection, or, a nucleic acid thathybridizes under stringent conditions to a nucleic acid sequence of theinvention. The overexpression can be effected by any means, e.g., use ofa high activity promoter, a dicistronic vector or by gene amplificationof the vector.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun 229:295-8),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Salmonella typhimurium and variousspecies within the genera Streptomyces and Staphylococcus, fungal cells,such as yeast, insect cells such as Drosophila S2 and Spodoptera Sf9,animal cells such as CHO, COS or Bowes melanoma and adenoviruses. Theselection of an appropriate host is within the abilities of thoseskilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981) and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150or more consecutive amino acids thereof can be synthetically produced byconventional peptide synthesizers. In other aspects, fragments orportions of the polypeptides may be employed for producing thecorresponding full-length polypeptide by peptide synthesis; therefore,the fragments may be employed as intermediates for producing thefull-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of the invention, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof using mRNAs transcribed from a DNA construct comprising apromoter operably linked to a nucleic acid encoding the polypeptide orfragment thereof. In some aspects, the DNA construct may be linearizedprior to conducting an in vitro transcription reaction. The transcribedmRNA is then incubated with an appropriate cell-free translationextract, such as a rabbit reticulocyte extract, to produce the desiredpolypeptide or fragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding enzymes of the invention, or modified nucleic acids ofthe invention, can be reproduced by amplification. Amplification canalso be used to clone or modify the nucleic acids of the invention.Thus, the invention provides amplification primer sequence pairs foramplifying nucleic acids of the invention. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and about the first(the 5′) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an enzymeactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesenzyme-encoding nucleic acids generated by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. The invention provides methods of making enzyme-encodingnucleic acids by amplification, e.g., polymerase chain reaction (PCR),using an amplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19)over a region of at least about 10, 20, 30, 40, 50, 60, 70, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550 or more, residues. The invention provides polypeptidescomprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to an exemplarypolypeptide of the invention. The extent of sequence identity (homology)may be determined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

Nucleic acid sequences of the invention can comprise at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivenucleotides of an exemplary sequence of the invention and sequencessubstantially identical thereto. Homologous sequences and fragments ofnucleic acid sequences of the invention can refer to a sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, or 50% homology to these sequences. Homology may be determinedusing any of the computer programs and parameters described herein,including FASTA version 3.0t78 with the default parameters. Homologoussequences also include RNA sequences in which uridines replace thethymines in the nucleic acid sequences of the invention. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences of the invention can berepresented in the traditional single character format (See the insideback cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co.,New York.) or in any other format which records the identity of thenucleotides in a sequence.

Various sequence comparison programs identified elsewhere in this patentspecification are particularly contemplated for use in this aspect ofthe invention. Protein and/or nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988;Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al.,Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., MethodsEnzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443, 1970, bythe search for similarity method of person & Lipman, Proc. Nat'l. Acad.Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project (J.Roach, http://weber.u.Washington.edu/˜roach/human_genome_progress2.html) (Gibbs, 1995). At least twenty-one other genomes have alreadybeen sequenced, including, for example, M. genitalium (Fraser et al.,1995), M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann etal., 1995), E. coli (Blattner et al., 1997) and yeast (S. cerevisiae)(Mewes et al., 1997) and D. melanogaster (Adams et al., 2000).Significant progress has also been made in sequencing the genomes ofmodel organism, such as mouse, C. elegans and Arabadopsis sp. Severaldatabases containing genomic information annotated with some functionalinformation are maintained by different organization and are accessiblevia the internet

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more in one aspect lessthan about 0.01 and most in one aspect less than about 0.001.

In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis in one aspect obtained from a protein or nucleic acid sequencedatabase. High-scoring segment pairs are in one aspect identified (L e.,aligned) by means of a scoring matrix, many of which are known in theart. In one aspect, the scoring matrix used is the BLOSUM62 matrix(Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff,Proteins 17:49-61, 1993). Less in one aspect, the PAM or PAM250 matricesmay also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matricesfor Detecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user.

In one aspect, the phrase “substantially identical” in the context oftwo nucleic acids or polypeptides, refers to two or more sequences thathave, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or morenucleotide or amino acid residue (sequence) identity, when compared andaligned for maximum correspondence, as measured using one of the knownsequence comparison algorithms or by visual inspection. In alternativeaspects, the substantial identity exists over a region of at least about50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, or thefull length of a gene or a transcript. In some aspects, the sequencesare substantially identical over the entire length of a coding region.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a nucleic acid or polypeptide sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

The polypeptides of the invention include the polypeptide sequences ofthe invention, e.g., the exemplary sequences of the invention, andsequences substantially identical thereto, and fragments of any of thepreceding sequences. Substantially identical, or homologous, polypeptidesequences refer to a polypeptide sequence having at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary sequence of the invention.

Homology may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutiveamino acids of the polypeptides of the invention. It will be appreciatedthat the polypeptide codes as set forth in amino acid sequences of theinvention, can be represented in the traditional single character formator three letter format (See the inside back cover of Stryer, Lubert.Biochemistry, 3rd Ed., W. H Freeman & Co., New York.) or in any otherformat which relates the identity of the polypeptides in a sequence.

A nucleic acid or polypeptide sequence of the invention can be stored,recorded and manipulated on any medium which can be read and accessed bya computer. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any of the presently known methods for recordinginformation on a computer readable medium to generate manufacturescomprising one or more of the nucleic acid sequences of the invention,one or more of the polypeptide sequences of the invention. Anotheraspect of the invention is a computer readable medium having recordedthereon at least 2, 5, 10, 15, or 20 or more nucleic acid sequences ofthe invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences of theinvention. Another aspect of the invention is a computer readable mediumhaving recorded thereon one or more of the polypeptide sequences of theinvention. Another aspect of the invention is a computer readable mediumhaving recorded thereon at least 2, 5, 10, 15, or 20 or more of thesequences as set forth above.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems which store and manipulate the sequenceinformation described herein. One example of a computer system 100 isillustrated in block diagram form in FIG. 7. As used herein, “a computersystem” refers to the hardware components, software components and datastorage components used to analyze a nucleotide sequence of a nucleicacid sequence of the invention, or a polypeptide sequence of theinvention. The computer system 100 typically includes a processor forprocessing, accessing and manipulating the sequence data. The processor105 can be any well-known type of central processing unit, such as, forexample, the Pentium III from Intel Corporation, or similar processorfrom Sun, Motorola, Compaq, AMD or International Business Machines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular aspect, the computer system 100 includes a processor105 connected to a bus which is connected to a main memory 115 (in oneaspect implemented as RAM) and one or more internal data storage devices110, such as a hard drive and/or other computer readable media havingdata recorded thereon. In some aspects, the computer system 100 furtherincludes one or more data retrieving device 118 for reading the datastored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some aspects, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence of the invention, or a polypeptide sequence of theinvention, (such as search tools, compare tools and modeling tools etc.)may reside in main memory 115 during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention, or a polypeptide sequence of the invention, stored on acomputer readable medium to a reference nucleotide or polypeptidesequence(s) stored on a computer readable medium. A “sequence comparisonalgorithm” refers to one or more programs which are implemented (locallyor remotely) on the computer system 100 to compare a nucleotide sequencewith other nucleotide sequences and/or compounds stored within a datastorage means. For example, the sequence comparison algorithm maycompare the nucleotide sequences of a nucleic acid sequence of theinvention, or a polypeptide sequence of the invention, stored on acomputer readable medium to reference sequences stored on a computerreadable medium to identify homologies or structural motifs.

FIG. 5 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention, or a polypeptide sequence of the invention, adata storage device having retrievably stored thereon referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention, or a polypeptide sequence of theinvention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs in the above described nucleicacid code a nucleic acid sequence of the invention, or a polypeptidesequence of the invention, or it may identify structural motifs insequences which are compared to these nucleic acid codes and polypeptidecodes. In some aspects, the data storage device may have stored thereonthe sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences of the invention, or the polypeptide sequences ofthe invention.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence of the invention, or apolypeptide sequence of the invention and a reference nucleotidesequence. The method including reading the nucleic acid code or thepolypeptide code and the reference nucleotide or polypeptide sequencethrough the use of a computer program which determines homology levelsand determining homology between the nucleic acid code or polypeptidecode and the reference nucleotide or polypeptide sequence with thecomputer program. The computer program may be any of a number ofcomputer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences of the invention, orthe polypeptide sequences of the invention through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 6 is a flow diagram illustrating one aspect of a process 250 in acomputer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is in one aspect in the single letter aminoacid code so that the first and sequence sequences can be easilycompared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of the invention,differs from a reference nucleic acid sequence at one or more positions.Optionally such a program records the length and identity of inserted,deleted or substituted nucleotides with respect to the sequence ofeither the reference polynucleotide or a nucleic acid sequence of theinvention. In one aspect, the computer program may be a program whichdetermines whether a nucleic acid sequence of the invention, contains asingle nucleotide polymorphism (SNP) with respect to a referencenucleotide sequence.

Accordingly, another aspect of the invention is a method for determiningwhether a nucleic acid sequence of the invention, differs at one or morenucleotides from a reference nucleotide sequence comprising the steps ofreading the nucleic acid code and the reference nucleotide sequencethrough use of a computer program which identifies differences betweennucleic acid sequences and identifying differences between the nucleicacid code and the reference nucleotide sequence with the computerprogram. In some aspects, the computer program is a program whichidentifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 6. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences of the invention and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other aspects the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence ofthe invention or a polypeptide sequence of the invention.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence of the invention, or apolypeptide sequence of the invention. In one aspect, the identifier maycomprise a program which identifies an open reading frame in a nucleicacid sequence of the invention.

FIG. 7 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic catalytic domains (CDs),or, active sites, helix-turn-helix motifs or other motifs known to thoseskilled in the art.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence of the invention, or apolypeptide sequence of the invention, comprising reading the nucleicacid code(s) or polypeptide code(s) through the use of a computerprogram which identifies features therein and identifying featureswithin the nucleic acid code(s) with the computer program. In oneaspect, computer program comprises a computer program which identifiesopen reading frames. The method may be performed by reading a singlesequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of the nucleic acidsequences of the invention, or the polypeptide sequences of theinvention, through the use of the computer program and identifyingfeatures within the nucleic acid codes or polypeptide codes with thecomputer program.

A nucleic acid sequence of the invention, or a polypeptide sequence ofthe invention, may be stored and manipulated in a variety of dataprocessor programs in a variety of formats. For example, a nucleic acidsequence of the invention, or a polypeptide sequence of the invention,may be stored as text in a word processing file, such as Microsoft WORD™or WORDPERFECT™ or as an ASCII file in a variety of database programsfamiliar to those of skill in the art, such as DB2™, SYBASE™, orORACLE™. In addition, many computer programs and databases may be usedas sequence comparison algorithms, identifiers, or sources of referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention, or a polypeptide sequence of theinvention. The following list is intended not to limit the invention butto provide guidance to programs and databases which are useful with thenucleic acid sequences of the invention, or the polypeptide sequences ofthe invention.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwents'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites (catalytic domains(CDs)), substrate binding sites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to an exemplary sequence ofthe invention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 orSEQ ID NO:19). The stringent conditions can be highly stringentconditions, medium stringent conditions and/or low stringent conditions,including the high and reduced stringency conditions described herein.In one aspect, it is the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid is withinthe scope of the invention, as discussed below.

In alternative aspects, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites (catalytic domains (CDs)) and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent) and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45 C in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10 C for the oligonucleotide probe. The membrane is thenexposed to auto-radiographic film for detection of hybridizationsignals.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows: 2×SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5 C from 68 C to 42 C in ahybridization buffer having a Na+ concentration of approximately 1M.Following hybridization, the filter may be washed with 2×SSC, 0.5% SDSat the temperature of hybridization. These conditions are considered tobe “moderate” conditions above 50 C and “low” conditions below 50 C. Aspecific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55 C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45 C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42 C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50 C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide A specificexample of “low stringency” hybridization conditions is when the abovehybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention. Forexample, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a nucleic acidsequence selected from the group consisting of one of the sequences ofthe invention, or fragments comprising at least about 10, 15, 20, 25,30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive basesthereof and the sequences complementary thereto. Homology may bemeasured using the alignment algorithm. For example, the homologouspolynucleotides may have a coding sequence which is a naturallyoccurring allelic variant of one of the coding sequences describedherein. Such allelic variants may have a substitution, deletion oraddition of one or more nucleotides when compared to the nucleic acidsof the invention.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a polypeptide ofthe invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof asdetermined using a sequence alignment algorithm (e.g., such as the FASTAversion 3.0t78 algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with an enzymeactivity or fragments thereof or for identifying genes or other nucleicacids encoding polypeptides having a chlorophyllase enzyme activity orenzymes involved in the catabolism of chlorophyll. In one aspect, theprobe comprises at least 10 consecutive bases of a nucleic acid of theinvention. Alternatively, a probe of the invention can be at least about5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 orabout 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of asequence as set forth in a nucleic acid of the invention. The probesidentify a nucleic acid by binding and/or hybridization. The probes canbe used in arrays of the invention, see discussion below, including,e.g., capillary arrays. The probes of the invention can also be used toisolate other nucleic acids or polypeptides.

The isolated nucleic acids of the invention, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of the invention, or the sequences complementary thereto mayalso be used as probes to determine whether a biological sample, such asa soil sample, contains an organism having a nucleic acid sequence ofthe invention or an organism from which the nucleic acid was obtained.In such procedures, a biological sample potentially harboring theorganism from which the nucleic acid was isolated is obtained andnucleic acids are obtained from the sample. The nucleic acids arecontacted with the probe under conditions which permit the probe tospecifically hybridize to any complementary sequences from which arepresent therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification—an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences of theinvention, may also be used in chromosome walking procedures to identifyclones containing genomic sequences located adjacent to the sequences ofthe invention. Such methods allow the isolation of genes which encodeadditional proteins from the host organism.

The isolated nucleic acids of the invention, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of the invention, or the sequences complementary thereto maybe used as probes to identify and isolate related nucleic acids. In someaspects, the related nucleic acids may be cDNAs or genomic DNAs fromorganisms other than the one from which the nucleic acid was isolated.For example, the other organisms may be related organisms. In suchprocedures, a nucleic acid sample is contacted with the probe underconditions which permit the probe to specifically hybridize to relatedsequences. Hybridization of the probe to nucleic acids from the relatedorganism is then detected using any of the methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 g denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 g denatured fragmented salmon sperm DNA, 50%formamide. The formulas for SSC and Denhardt's solutions are listed inSambrook et al., supra.

In one aspect, hybridization is conducted by adding the detectable probeto the prehybridization solutions listed above. Where the probecomprises double stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T. In one aspect, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. In oneaspect, for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

Inhibiting Expression of Enzymes

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g., nucleic acidsencoding polypeptides having an enzyme activity involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity.Antisense sequences are capable of inhibiting the transport, splicing ortranscription of enzyme-encoding genes. The inhibition can be effectedthrough the targeting of genomic DNA or messenger RNA. The transcriptionor function of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One particularly useful set of inhibitorsprovided by the present invention includes oligonucleotides which areable to either bind gene or message, in either case preventing orinhibiting the production or function of the desired enzyme. Theassociation can be through sequence specific hybridization. Anotheruseful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of nucleic acids encoding polypeptides havingan enzyme activity involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity. The oligonucleotide can haveenzyme activity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. A poolof many different such oligonucleotides can be screened for those withthe desired activity. Thus, the invention provides various compositionsfor the inhibition of enzyme expression on a nucleic acid and/or proteinlevel, e.g., antisense, iRNA and ribozymes comprising nucleic acidsequences of the invention and antibodies of the invention.

Inhibition of expression of nucleic acids encoding polypeptides havingan enzyme activity involved in chlorophyll catabolism or having anesterase (e.g., chlorophyllase) activity can have a variety ofindustrial applications. For example, compositions of the invention forthe inhibition of enzyme expression (e.g., antisense, iRNA, ribozymes,antibodies) can be used as pharmaceutical compositions, e.g., asanti-pathogen agents or in other therapies, e.g., where the inhibitedenzyme has an undesired, deleterious or toxic effect.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingenzyme message or a gene which can inhibit a target gene or message to,e.g., inhibit a polypeptide involved in chlorophyll catabolism or havingan esterase (e.g., chlorophyllase) activity by targeting mRNA.Strategies for designing antisense oligonucleotides are well describedin the scientific and patent literature, and the skilled artisan candesign such oligonucleotides using the novel reagents of the invention.For example, gene walking/RNA mapping protocols to screen for effectiveantisense oligonucleotides are well known in the art, see, e.g., Ho(2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay,which is based on standard molecular techniques to provide an easy andreliable method for potent antisense sequence selection. See also Smith(2000) Eur. J. Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding message or genesencoding polypeptides of the invention, or, encoding polypeptidesinvolved in chlorophyll catabolism or having an esterase (e.g.,chlorophyllase) activity. These ribozymes can inhibit activity by, e.g.,targeting mRNA. Strategies for designing ribozymes and selecting theenzyme-specific antisense sequence for targeting are well described inthe scientific and patent literature, and the skilled artisan can designsuch ribozymes using the novel reagents of the invention. Ribozymes actby binding to a target RNA through the target RNA binding portion of aribozyme which is held in close proximity to an enzymatic portion of theRNA that cleaves the target RNA. Thus, the ribozyme recognizes and bindsa target RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cleave and inactivate the targetRNA. Cleavage of a target RNA in such a manner will destroy its abilityto direct synthesis of an encoded protein if the cleavage occurs in thecoding sequence. After a ribozyme has bound and cleaved its RNA target,it can be released from that RNA to bind and cleave new targetsrepeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a sequence of the invention. TheRNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAican inhibit expression of a nucleic acid encoding a polypeptide involvedin chlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity, e.g., as described herein. In one aspect, the RNAi is about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides inlength. While the invention is not limited by any particular mechanismof action, the RNAi can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA),mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi). A possible basic mechanism behind RNAiis the breaking of a double-stranded RNA (dsRNA) matching a specificgene sequence into short pieces called short interfering RNA, whichtrigger the degradation of mRNA that matches its sequence. In oneaspect, the RNAi's of the invention are used in gene-silencingtherapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. Inone aspect, the invention provides methods to selectively degrade RNAusing the RNAi's of the invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the RNAi molecules of theinvention can be used to generate a loss-of-function mutation in a cell,an organ or an animal. Methods for making and using RNAi molecules forselectively degrade RNA are well known in the art, see, e.g., U.S. Pat.Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a polypeptide involved inchlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity, e.g., enzymes described herein. These methods can be repeatedor used in various combinations to generate polypeptides involved inchlorophyll catabolism or having an esterase (e.g., chlorophyllase)activity having an altered or different activity or an altered ordifferent stability from that of an enzyme encoded by the templatenucleic acid. These methods also can be repeated or used in variouscombinations, e.g., to generate variations in gene/message expression,message translation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfate, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis™ (GSSM™), synthetic ligation reassembly(SLR), recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation, and/or a combination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar (1988) “Total synthesis and expression of a gene for thea-subunit of bovine rod outer segment guanine nucleotide-binding protein(transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985)“Cassette mutagenesis: an efficient method for generation of multiplemutations at defined sites” Gene 34:315-323; and Grundstrom et al.(1985) “Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis” Nucl. Acids Res. 13: 3305-3316), double-strand breakrepair (Mandecki (1986); Arnold (1993) “Protein engineering for unusualenvironments” Current Opinion in Biotechnology 4:450-455.“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177-7181). Additional details on many of the abovemethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g., GeneSite Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),or a combination thereof are used to modify the nucleic acids of theinvention to generate polypeptides involved in chlorophyll catabolism orhaving an esterase (e.g., chlorophyllase) activity with new or alteredproperties (e.g., activity under highly acidic or alkaline conditions,high or low temperatures, and the like). Polypeptides encoded by themodified nucleic acids can be screened for an activity before testingfor glucan or other polysaccharide hydrolysis or other activity. Anytesting modality or protocol can be used, e.g., using a capillary arrayplatform. See, e.g., U.S. Pat. Nos. 6,361,974; 6,280,926; 5,939,250.

Gene Site Saturation Mutagenesis (GSSM)

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., anucleic acid of the invention, so as to generate a set of progenypolypeptides in which a full range of single amino acid substitutions isrepresented at each amino acid position, e.g., an amino acid residue inan enzyme active site (catalytic domains (CDs)) or ligand binding sitetargeted to be modified. These oligonucleotides can comprise acontiguous first homologous sequence, a degenerate N,N,G/T sequence,and, optionally, a second homologous sequence. The downstream progenytranslational products from the use of such oligonucleotides include allpossible amino acid changes at each amino acid site along thepolypeptide, because the degeneracy of the N,N,G/T sequence includescodons for all 20 amino acids. In one aspect, one such degenerateoligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) isused for subjecting each original codon in a parental polynucleotidetemplate to a full range of codon substitutions. In another aspect, atleast two degenerate cassettes are used—either in the sameoligonucleotide or not, for subjecting at least two original codons in aparental polynucleotide template to a full range of codon substitutions.For example, more than one N,N,G/T sequence can be contained in oneoligonucleotide to introduce amino acid mutations at more than one site.This plurality of N,N,G/T sequences can be directly contiguous, orseparated by one or more additional nucleotide sequence(s). In anotheraspect, oligonucleotides serviceable for introducing additions anddeletions can be used either alone or in combination with the codonscontaining an N,N,G/T sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,polypeptides of the invention involved in chlorophyll catabolism orhaving an esterase (e.g., chlorophyllase) activity) molecules such thatall 20 natural amino acids are represented at the one specific aminoacid position corresponding to the codon position mutagenized in theparental polynucleotide (other aspects use less than all 20 naturalcombinations). The 32-fold degenerate progeny polypeptides generatedfrom each saturation mutagenesis reaction vessel can be subjected toclonal amplification (e.g. cloned into a suitable host, e.g., E. colihost, using, e.g., an expression vector) and subjected to expressionscreening. When an individual progeny polypeptide is identified byscreening to display a favorable change in property (when compared tothe parental polypeptide, such as increased glucan hydrolysis activityunder alkaline or acidic conditions), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (Gene Site Saturation Mutagenesis™ (GSSM™)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and in one aspect but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where the N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changesand point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is in one aspect everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(in one aspect a subset totaling from 15 to 100,000) to mutagenesis. Inone aspect, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations can beintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette is in oneaspect about 1-500 bases in length) in defined polynucleotide sequenceto be mutagenized (wherein the sequence to be mutagenized is in oneaspect from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., enzymes of the invention, withnew or altered properties. SLR is a method of ligating oligonucleotidefragments together non-stochastically. This method differs fromstochastic oligonucleotide shuffling in that the nucleic acid buildingblocks are not shuffled, concatenated or chimerized randomly, but ratherare assembled non-stochastically. See, e.g., U.S. patent applicationSer. No. 09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are in one aspect shared by atleast two of the progenitor templates. The demarcation points canthereby be used to delineate the boundaries of oligonucleotide buildingblocks to be generated in order to rearrange the parentalpolynucleotides. The demarcation points identified and selected in theprogenitor molecules serve as potential chimerization points in theassembly of the final chimeric progeny molecules. A demarcation pointcan be an area of homology (comprised of at least one homologousnucleotide base) shared by at least two parental polynucleotidesequences. Alternatively, a demarcation point can be an area of homologythat is shared by at least half of the parental polynucleotidesequences, or, it can be an area of homology that is shared by at leasttwo thirds of the parental polynucleotide sequences. Even more in oneaspect a serviceable demarcation points is an area of homology that isshared by at least three fourths of the parental polynucleotidesequences, or, it can be shared by at almost all of the parentalpolynucleotide sequences. In one aspect, a demarcation point is an areaof homology that is shared by all of the parental polynucleotidesequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedin one aspect comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly, that is somewhat related to stochasticshuffling, save that the nucleic acid building blocks are not shuffledor concatenated or chimerized randomly, but rather are assemblednon-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thepolypeptides of the present invention can be mutagenized in accordancewith the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofthe invention) are aligned in order to select one or more demarcationpoints, which demarcation points can be located at an area of homology.The demarcation points can be used to delineate the boundaries ofnucleic acid building blocks to be generated. Thus, the demarcationpoints identified and selected in the progenitor molecules serve aspotential chimerization points in the assembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates and in one aspect at almost all of theprogenitor templates. Even more in one aspect still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one aspect, the gene reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that the gene reassembly processis performed systematically, for example to generate a systematicallycompartmentalized library, with compartments that can be screenedsystematically, e.g., one by one. In other words the invention providesthat, through the selective and judicious use of specific nucleic acidbuilding blocks, coupled with the selective and judicious use ofsequentially stepped assembly reactions, an experimental design can beachieved where specific sets of progeny products are made in each ofseveral reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated in one aspect comprise a library offinalized chimeric nucleic acid molecules having an overall assemblyorder that is chosen by design. In a particularly aspect, such agenerated library is comprised of greater than 10³ to greater than10¹⁰⁰⁰ different progeny molecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutagenesis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). In one aspect, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made, intron-containing gene and a nucleic acid, whichserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which in one aspecthas two ligatable ends. The two ligatable ends on each nucleic acidbuilding block may be two blunt ends (i.e. each having an overhang ofzero nucleotides), or in one aspect one blunt end and one overhang, ormore in one aspect still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherexemplary size ranges are also provided, which have lower limits of from1 by to 10,000 bp (including every integer value in between) and upperlimits of from 2 by to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. In one aspect the codon degeneracy isintroduced using the site-saturation mutagenesis described herein, usingone or more N,N,G/T cassettes or alternatively using one or more N,N,Ncassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,enzymes or antibodies of the invention, with new or altered properties.Optimized directed evolution is directed to the use of repeated cyclesof reductive reassortment, recombination and selection that allow forthe directed molecular evolution of nucleic acids through recombination.Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide in one aspect includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide in one aspectincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is in one aspect performed in MATLAB™ (The Mathworks, Natick,Mass.) a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new phenotype is identified, re-isolated (e.g., usinga nucleic acid of the invention), again modified, re-tested foractivity. This process can be iteratively repeated until a desiredphenotype is engineered. For example, an entire biochemical anabolic orcatabolic pathway can be engineered into a cell, including, e.g., new oraltered biosynthetic or (e.g., chlorophyll) degradative pathway.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new or altered biosyntheticor (e.g., chlorophyll) degradative pathway phenotype), it can be removedas a variable by synthesizing larger parental oligonucleotides thatinclude the sequence to be removed. Since incorporating the sequencewithin a larger sequence prevents any crossover events, there will nolonger be any variation of this sequence in the progeny polynucleotides.This iterative practice of determining which oligonucleotides are mostrelated to the desired trait, and which are unrelated, allows moreefficient exploration all of the possible protein variants that might beprovide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,enzymes and the like. In vivo shuffling can be performed utilizing thenatural property of cells to recombine multimers. While recombination invivo has provided the major natural route to molecular diversity,genetic recombination remains a relatively complex process thatinvolves 1) the recognition of homologies; 2) strand cleavage, strandinvasion, and metabolic steps leading to the production of recombinantchiasma; and finally 3) the resolution of chiasma into discreterecombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19and combinations thereof) into a suitable host cell. The regions ofpartial sequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNaseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

-   1) The use of vectors only stably maintained when the construct is    reduced in complexity.-   2) The physical recovery of shortened vectors by physical    procedures. In this case, the cloning vector would be recovered    using standard plasmid isolation procedures and size fractionated on    either an agarose gel, or column with a low molecular weight cut off    utilizing standard procedures.-   3) The recovery of vectors containing interrupted genes which can be    selected when insert size decreases.-   4) The use of direct selection techniques with an expression vector    and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution and the like)and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5A-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid sequences of the invention. The inventionalso provides additional methods for isolating polypeptides of theinvention. In one aspect, the invention provides for variants of codingsequences (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G.F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids areevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D. C., PNAS, USA,89:7811-7815, 1992.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S. and Youvan, D.C., Biotechnology Research, 11:1548-1552,1993. Random and site-directed mutagenesis are described in Arnold,F.H., Current Opinion in Biotechnology, 4:450-455, 1993.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides of the invention may be variants inwhich one or more of the amino acid residues of the polypeptides of thesequences of the invention are substituted with a conserved ornon-conserved amino acid residue (in one aspect a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code.

The invention provides alternative embodiments of the polypeptides ofthe invention (and the nucleic acids that encode them) comprising atleast one conservative amino acid substitution, as discussed herein(e.g., conservative amino acid substitutions are those that substitute agiven amino acid in a polypeptide by another amino acid of likecharacteristics). The invention provides polypeptides (and the nucleicacids that encode them) wherein any, some or all amino acids residuesare substituted by another amino acid of like characteristics, e.g., aconservative amino acid substitution.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue. In alternativeaspects, these conservative substitutions can also be syntheticequivalents of these amino acids.

Other variants are those in which one or more of the amino acid residuesof a polypeptide of the invention includes a substituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide.

In some aspects, the fragments, derivatives and analogs retain the samebiological function or activity as the polypeptides of the invention. Inother aspects, the fragment, derivative, or analog includes aproprotein, such that the fragment, derivative, or analog can beactivated by cleavage of the proprotein portion to produce an activepolypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying nucleic acids encodingpolypeptides involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity by modifying codon usage. In one aspect,the invention provides methods for modifying codons in a nucleic acidencoding a polypeptide to increase or decrease its expression in a hostcell. The invention also provides nucleic acids encoding polypeptidesinvolved in chlorophyll catabolism or having an esterase (e.g.,chlorophyllase) activity modified to increase its expression in a hostcell, enzymes so modified, and methods of making the modifiedpolypeptides involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity. The method comprises identifying a“non-preferred” or a “less preferred” codon in enzyme-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as Streptomyces, Lactobacillusgasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus sp.,Bacillus subtilis, Bacillus cereus. Exemplary host cells also includeeukaryotic organisms, e.g., various yeast, such as Saccharomyces sp.,including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillusniger, and mammalian cells and cell lines and insect cells and celllines. Thus, the invention also includes nucleic acids and polypeptidesoptimized for expression in these organisms and species, e.g., thenucleic acids of the invention are codon-optimized for expression in ahost cell, e.g., a Pichia sp., e.g., P. pastoris, a Saccharomyces sp.,or a Bacillus sp., a Streptomyces sp., and the like.

For example, the codons of a nucleic acid encoding a polypeptide of theinvention or a similar enzyme isolated from a bacterial cell aremodified such that the nucleic acid (encoding the enzyme) is optimallyexpressed in a bacterial cell different from the bacteria from which theenzyme (e.g., a polypeptide of the invention) was derived, a yeast, afungi, a plant cell, an insect cell or a mammalian cell. Methods foroptimizing codons are well known in the art, see, e.g., U.S. Pat. No.5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998)Protein Expr. Purif. 12:185-188; Narum (2001) Infect. Immun69:7250-7253. See also Narum (2001) Infect. Immun 69:7250-7253,describing optimizing codons in mouse systems; Outchkourov (2002)Protein Expr. Purif. 24:18-24, describing optimizing codons in yeast;Feng (2000) Biochemistry 39:15399-15409, describing optimizing codons inE. coli; Humphreys (2000) Protein Expr. Purif. 20:252-264, describingoptimizing codon usage that affects secretion in E. coli; Gao (2004)Biotechnol Prog. 20:443-448, describing “UpGene”, an application of aweb-based DNA codon optimization algorithm.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The invention also providesmethods of making and using these transgenic non-human animals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyenzyme activity, or, as models to screen for agents that change theenzyme activity in vivo. The coding sequences for the polypeptides to beexpressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing and enzyme of the invention, or, a fusion proteincomprising an enzyme of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a polypeptide involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity), anexpression cassette or vector or a transfected or transformed cell ofthe invention. The invention also provides plant products, e.g., oils,seeds, leaves, extracts and the like, comprising a nucleic acid and/or apolypeptide of the invention. The transgenic plant can be dicotyledonous(a dicot) or monocotyledonous (a monocot). The invention also providesmethods of making and using these transgenic plants and seeds. Thetransgenic plant or plant cell expressing a polypeptide of the inventionmay be constructed in accordance with any method known in the art. See,for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's endogenous transcriptional and/or translational controlelements regulate the activity of the introduced nucleic acid, whetherit be integrated or episomal. The invention also provides “knockoutplants” where insertion of gene sequence by, e.g., homologousrecombination, has disrupted the expression of the endogenous gene.Means to generate “knockout” plants are well-known in the art, see,e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995)Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of a polypeptide of theinvention or a homologous enzyme in the host. This can change enzyme(e.g., chlorophyllase) activity or biosynthetic pathway product (achlorophyll degradative pathway) in the plant. Alternatively, an enzymeor nucleic acid of the invention can be used in production of atransgenic plant to produce a compound not naturally produced by thatplant. This can lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice. Nucleicacids, e.g., expression constructs, can also be introduced in to plantcells using recombinant viruses. Plant cells can be transformed usingviral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering or seeding behavior isaltered) can be enhanced when both parental plants express thepolypeptides of the invention. The desired effects can be passed tofuture plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., enzymes or antibody)of the invention. For example, see Palmgren (1997) Trends Genet. 13:348;Chong (1997) Transgenic Res. 6:289-296 (producing human milk proteinbeta-casein in transgenic potato plants using an auxin-inducible,bidirectional mannopine synthase (mas 1′,2′) promoter with Agrobacteriumtumefaciens-mediated leaf disc transformation methods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated, synthetic or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention, e.g., proteinshaving a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18 or SEQ ID NO:20.

In one aspect, a polypeptide of the invention has an esterase activity,such as a chlorophyllase (a chlase) activity, or, has an enzyme activitycomprising enzymatic modification of a chlorophyll molecule, e.g.,wherein the enzymatic modification comprises catabolism of thechlorophyll molecule. In one aspect, the esterase activity comprises achlorophyll chlorophyllido-hydrolyase activity.

Another aspect of the invention provides an isolated, synthetic orrecombinant polypeptide or peptide including at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or moreconsecutive bases of a polypeptide or peptide sequence of the invention,sequences substantially identical thereto, and the sequencescomplementary thereto. The peptide can be, e.g., an immunogenicfragment, a motif (e.g., a binding site), a signal sequence, a preprosequence or a catalytic domains (CDs) or active site.

The invention also provides chimeric polypeptides (and the nucleic acidsencoding them) comprising at least two enzymes of the invention orsubsequences thereof, e.g., active sites, or catalytic domains (CDs). Achimeric protein of the invention (e.g., a fusion protein, or, otherheterodimer, e.g., two domains joined by other means, e.g., a linker,or, electrostatically) can comprise one polypeptide (e.g., active siteor catalytic domain peptide) of the invention and another polypeptide(e.g., active site or catalytic domain peptide) of the invention orother polypeptide. For example, a chimeric protein of the invention canhave any activity of a polypeptide involved chlorophyll catabolism orhaving an esterase (e.g., chlorophyllase) activity, e.g., as describedherein. In one aspect the chimeric protein of the invention comprises afusion of domains, e.g., a single domain can exhibit one or anycombination of activities.

The polypeptides of the invention include enzymes in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude enzymes inactive for other reasons, e.g., before “activation” bya post-translational processing event, e.g., an endo- or exo-peptidaseor proteinase action, a phosphorylation event, an amidation, aglycosylation or a sulfation, a dimerization event, and the like. Thepolypeptides of the invention include all active forms, including activesubsequences, e.g., catalytic domains or active sites, of the enzymes.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences, prepro sequences and catalytic domains(e.g., “active sites”) comprising sequences of the invention.

The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700 or more residues. Polypeptides of the invention can also beshorter than the full length of exemplary polypeptides. In alternativeaspects, the invention provides polypeptides (peptides, fragments)ranging in size between about 5 and the full length of a polypeptide,e.g., an enzyme, of the invention; exemplary sizes being of about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, ormore residues, e.g., contiguous residues of an exemplary enzymes of theinvention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, enzyme active sites (e.g., “catalyticdomains” of enzymes of the invention), binding sites of enzymes of theinvention, signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A.K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered from an exemplarypolypeptide of the invention. In one aspect, a mimetic composition isused in a composition, cell system or process of the invention (e.g., ahost cell having a plasmid expressing at least one enzyme of theinvention).

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, inone aspect under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention includes polypeptides of the invention with and withoutsignal. The polypeptide comprising a signal sequence of the inventioncan be a polypeptide of the invention or another polypeptide.

The invention includes immobilized polypeptides of the invention,including enzymes, antibodies and fragments thereof. The inventionprovides methods for inhibiting polypeptide activity, e.g., usingdominant negative mutants or antibodies of the invention. The inventionincludes heterocomplexes, e.g., fusion proteins, heterodimers, etc.,comprising the enzymes of the invention.

Polypeptides of the invention can have enzyme activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativeenzyme preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, enzyme variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofenzyme variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify enzyme modulators, e.g., activators or inhibitors of enzymeactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to enzyme assays to determine their ability to inhibitsubstrate cleavage Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Enzymeinhibitors can be combined to increase the spectrum of activity.

The invention also provides methods of discovering a new enzymes havingsimilar activity to an enzyme of the invention using the nucleic acids,polypeptides and antibodies of the invention. In one aspect, phagemidlibraries are screened for expression-based discovery of a new enzyme.In another aspect, lambda phage libraries are screened forexpression-based discovery of a new enzyme. Screening of the phage orphagemid libraries can allow the detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of phage or phagemidlibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

Another aspect of the invention is an isolated or purified polypeptidecomprising the sequence of one of the invention, or fragments comprisingat least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. As discussed above, such polypeptidesmay be obtained by inserting a nucleic acid encoding the polypeptideinto a vector such that the coding sequence is operably linked to asequence capable of driving the expression of the encoded polypeptide ina suitable host cell. For example, the expression vector may comprise apromoter, a ribosome binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying expression.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or more than about 95% sequence identity (homology) to one of thepolypeptides of the invention, or a fragment comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive aminoacids thereof. Sequence identity (homology) may be determined using anyof the programs described above which aligns the polypeptides orfragments being compared and determines the extent of amino acididentity or similarity between them. It will be appreciated that aminoacid equivalence, or identity, or “homology,” includes conservativeamino acid substitutions such as those described above.

The polypeptides or fragments having homology to one of the polypeptidesof the invention, or a fragment comprising at least about 5, 10, 15, 20,25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof maybe obtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byactivity assays, gel electrophoresis and/or microsequencing. Thesequence of the prospective homologous polypeptide or fragment can becompared to one of the polypeptides of the invention, or a fragmentcomprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or150 consecutive amino acids thereof using any of the programs describedabove.

Another aspect of the invention is an assay for identifying fragments orvariants of the invention, which retain the enzymatic function of thepolypeptides of the invention. For example the fragments or variants ofpolypeptides of the invention may be used to catalyze biochemicalreactions, which indicate that the fragment or variant retains theenzymatic activity of a polypeptide of the invention.

The assay for determining if fragments of variants retain the enzymaticactivity of the polypeptides of the invention includes the steps of:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction and detecting either a decrease in the level of substrate or anincrease in the level of the specific reaction product of the reactionbetween the polypeptide and substrate.

The polypeptides of the invention or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof may be used in a variety of applications. For example, thepolypeptides or fragments thereof may be used to catalyze biochemicalreactions. In accordance with one aspect of the invention, there isprovided a process for utilizing the polypeptides of the invention orpolynucleotides encoding such polypeptides for hydrolyzing esterlinkages. In such procedures, a substance containing an ester linkage(e.g., a chlorophyll) is contacted with one of the polypeptides of theinvention, or sequences substantially identical thereto under conditionswhich facilitate the hydrolysis of the ester linkage.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular aspect, the invention provides a method for modifyingsmall molecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

Signal Sequences, Prepro, Binding Domains and Catalytic Domains

The invention provides enzyme signal sequences (e.g., signal peptides(SPs)), prepro domains, binding domains and catalytic domains (CDs)(e.g., active sites). The SPs, prepro domains and/or CDs of theinvention can be isolated or recombinant peptides or can be part of afusion protein, e.g., as a heterologous domain in a chimeric protein.The invention provides nucleic acids encoding these catalytic domains(CDs), prepro domains and signal sequences (SPs, e.g., a peptide havinga sequence comprising/consisting of amino terminal residues of apolypeptide of the invention). In one aspect, the invention provides asignal sequence comprising a peptide comprising/consisting of a sequenceas set forth in residues 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, 1to 47, 1 to 48, 1 to 49, 1 to 50, 1 to 51, or 1 to 52 or more, of apolypeptide of the invention.

In one aspect, the invention also provides chimeric polypeptides (andthe nucleic acids encoding them) comprising at least two enzymes of theinvention or subsequences thereof, e.g., catalytic domains (CDs) oractive sites. For example, a chimeric protein of the invention can haveany combination of activities. In one aspect the chimeric protein of theinvention comprises a fusion of domains, e.g., a single domain canexhibit one or any combination of activities (e.g., as a recombinantchimeric protein).

The invention also provides isolated, synthetic or recombinant signalsequences comprising/consisting of a signal sequence of the invention,e.g., exemplary signal sequences as set forth in Table 1, below, andpolypeptides comprising these signal sequences. The polypeptide can beanother enzyme of the invention, or another type of enzyme orpolypeptide. For example, to aid in reading Table 1, the inventionprovides an isolated, synthetic or recombinant signal sequence as setforth by the amino terminal amino acid residues 1 to 21(“NH₂-MSRVCLPLTLTLALTLSARA”) of SEQ ID NO: 2, encoded, e.g., by SEQ IDNO:1, etc.:

TABLE 1 Signal sequence position SEQ (AA = ID Amino NO: Acid)Signal sequence 1, 2 AA1-20 MSRVCLPLTLTLALTLSARA 11, 12 13, 14 15, 1617, 18 AA1-25 MKKYKTGLVLSGGGTRGFAHLGVIA 19, 20 3, 4 5, 6 AA1-25MRRIVFLYILALLCVSCANRNPSVS 7, 8 AA1-51 MTRKKIGLALSGGAARGFAHLGVLKVFAEHGIPVDFVAGTSAGSFAGAAFA 9, 10 AA1-23 MFNKALPAAAAVAGLFLSTSAMA

The signal sequences (SPs) and/or prepro sequences of the invention canbe isolated peptides, or, sequences joined to another enzyme of theinvention, or a heterologous protein, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisingsignal sequences of the invention. In one aspect, polypeptidescomprising signal sequences SPs and/or prepro of the invention comprisesequences heterologous to enzymes of the invention (e.g., a fusionprotein comprising an SP and/or prepro of the invention and/or sequencesfrom another protein). In one aspect, the invention provides an enzymeof the invention with heterologous SPs and/or prepro sequences, e.g.,sequences with a yeast signal sequence. Enzymes of the invention cancomprise a heterologous SP and/or prepro in a vector, e.g., a pPICseries vector (Invitrogen, Carlsbad, Calif.). In one aspect, SPs and/orprepro sequences of the invention are identified followingidentification of novel polypeptides. The pathways by which proteins aresorted and transported to their proper cellular location are oftenreferred to as protein targeting pathways. One of the most importantelements in all of these targeting systems is a short amino acidsequence at the amino terminus of a newly synthesized polypeptide calledthe signal sequence. This signal sequence directs a protein to itsappropriate location in the cell and is removed during transport or whenthe protein reaches its final destination. Most lysosomal, membrane, orsecreted proteins have an amino-terminal signal sequence that marks themfor translocation into the lumen of the endoplasmic reticulum. More than100 signal sequences for proteins in this group have been determined.The signal sequences can vary in length from 13 to 36 amino acidresidues. Various methods of recognition of signal sequences are knownto those of skill in the art. For example, in one aspect, novel signalpeptides are identified by a method referred to as SignalP. SignalP usesa combined neural network which recognizes both signal peptides andtheir cleavage sites. (Nielsen, et al., “Identification of prokaryoticand eukaryotic signal peptides and prediction of their cleavage sites.”Protein Engineering, vol. 10, no. 1, p. 1-6 (1997).

It should be understood that in some aspects an enzyme of the inventionmay not have SPs and/or prepro sequences, or one or more “domains.” Inone aspect, the invention provides an enzyme of the invention lackingall or part of an SP and/or a prepro domain. In one aspect, theinvention provides a nucleic acid sequence encoding a signal sequence(SP) and/or prepro from one enzyme of the invention operably linked to anucleic acid sequence of a different enzyme of the invention or,optionally, a signal sequence (SPs) and/or prepro domain from adifferent type of protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toenzymes of the invention) with an SP, prepro domain and/or CD. Thesequence to which the SP, prepro domain and/or CD are not naturallyassociated can be on the SP's, prepro domain and/or CD's amino terminalend, carboxy terminal end, and/or on both ends of the SP and/or CD. Inone aspect, the invention provides an isolated or recombinantpolypeptide comprising (or consisting of) a polypeptide comprising asignal sequence (SP), prepro domain and/or catalytic domain (CD) of theinvention with the proviso that it is not associated with any sequenceto which it is naturally associated. Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

Hybrid (Chimeric) Enzymes and Peptide Libraries

In one aspect, the invention provides hybrid enzymes of the inventionand fusion proteins, including peptide libraries, comprising sequencesof the invention. The peptide libraries of the invention can be used toisolate peptide modulators (e.g., activators or inhibitors) of targets,such as enzyme of the invention, their substrates, etc. The peptidelibraries of the invention can be used to identify formal bindingpartners of targets, such as ligands, e.g., cytokines, hormones and thelike. In one aspect, the invention provides chimeric proteins comprisinga signal sequence (SP), prepro domain and/or catalytic domain (CD) ofthe invention or a combination thereof and a heterologous sequence (seeabove).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of enzymes of the invention and other peptides, including knownand random peptides. They can be fused in such a manner that thestructure of a polypeptide is not significantly perturbed and thepeptide is metabolically or structurally conformationally stabilized.This allows the creation of a peptide library that is easily monitoredboth for its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of enzymes of the invention. In one aspect, the variants ofthe invention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed enzyme variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofchlorophyll hydrolysis, as described in Example 1, below. In alternativeaspects, amino acid substitutions can be single residues; insertions canbe on the order of from about 1 to 20 amino acids, although considerablylarger insertions can be done. Deletions can range from about 1 to about20, 30, 40, 50, 60, 70 residues or more. To obtain a final derivativewith the optimal properties, substitutions, deletions, insertions or anycombination thereof may be used. Generally, these changes are done on afew amino acids to minimize the alteration of the molecule. However,larger changes may be tolerated in certain circumstances.

The invention provides polypeptides, e.g., enzymes, of the inventionwhere the structure of the polypeptide backbone, the secondary or thetertiary structure, e.g., an alpha-helical or beta-sheet structure, hasbeen modified. In one aspect, the charge or hydrophobicity has beenmodified. In one aspect, the bulk of a side chain has been modified.Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative. For example,substitutions can be made which more significantly affect: the structureof the polypeptide backbone in the area of the alteration, for example aalpha-helical or a beta-sheet structure; a charge or a hydrophobic siteof the molecule, which can be at an active site; or a side chain. Theinvention provides substitutions in polypeptide of the invention where(a) a hydrophilic residues, e.g. seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g. lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine. The variants can exhibitthe same qualitative biological activity as enzymes of the invention,although variants can be selected to modify the characteristics of theenzyme as needed.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterm “polypeptide” as used herein, refers to amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres and may contain modified amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment of aphosphatidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pegylation, glucanhydrolase processing, phosphorylation, prenylation, racemization,selenoylation, sulfation and transfer-RNA mediated addition of aminoacids to protein such as arginylation. (See Creighton, T. E.,Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)). The peptides and polypeptides of the invention also include all“mimetic” and “peptidomimetic” forms, as described in further detail,below.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site (catalytic domains (CDs)) of the molecule andprovided that the polypeptide essentially retains its functionalproperties. A conservative amino acid substitution, for example,substitutes one amino acid for another of the same class (e.g.,substitution of one hydrophobic amino acid, such as isoleucine, valine,leucine, or methionine, for another, or substitution of one polar aminoacid for another, such as substitution of arginine for lysine, glutamicacid for aspartic acid or glutamine for asparagine). One or more aminoacids can be deleted, for example, from a polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for enzymebiological activity can be removed. Modified polypeptide sequences ofthe invention can be assayed for biological (e.g., enzymatic, orbinding) activity by any number of methods, including contacting themodified polypeptide sequence with an enzyme substrate and determiningwhether the modified polypeptide decreases the amount of specificsubstrate in the assay or increases the bioproducts of the enzymaticreaction of a functional polypeptide with the substrate.

“Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In alternative aspects, two amino acid sequencesare “substantially the same” or “substantially homologous” if they haveat least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity. Fragments which have different three dimensional structures asthe naturally occurring protein are also included; e.g., a “pro-form”molecule, such as a low activity proprotein that can be modified bycleavage to produce a mature enzyme with significantly higher activity.

In one aspect, enzymes of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, enzymes can be fused to a random peptide to form a fusionpolypeptide. By “fused” or “operably linked” herein is meant that therandom peptide and the enzyme are linked together, in such a manner asto minimize the disruption to the stability of the enzyme structure,e.g., it retains activity. The fusion polypeptide (or fusionpolynucleotide encoding the fusion polypeptide) can comprise furthercomponents as well, including multiple peptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridenzymes of the invention). In one aspect, the original polynucleotidesencode biologically active polypeptides. The method of the inventionproduces new hybrid polypeptides by utilizing cellular processes whichintegrate the sequence of the original polynucleotides such that theresulting hybrid polynucleotide encodes a polypeptide demonstratingactivities derived from the original biologically active polypeptides.For example, the original polynucleotides may encode a particular enzymefrom different microorganisms. An enzyme encoded by a firstpolynucleotide from one organism or variant may, for example, functioneffectively under a particular environmental condition, e.g. highsalinity. An enzyme encoded by a second polynucleotide from a differentorganism or variant may function effectively under a differentenvironmental condition, such as extremely high temperatures. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme which exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide may function effectively underenvironmental conditions shared by each of the enzymes encoded by thefirst and second polynucleotides, e.g., high salinity and extremetemperatures.

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding enzyme activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized enzyme activities obtained from each of theoriginal enzymes, i.e. the type of bond on which the enzyme acts and thetemperature at which the enzyme functions. Thus, for example, the enzymemay be screened to ascertain those chemical functionalities whichdistinguish the hybrid enzyme from the original enzymes, such assubstrate specificity, or temperature, pH or salt concentration at whichthe hybrid polypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Such enzymes may function at temperaturesabove 100° C. in terrestrial hot springs and deep sea thermal vents, attemperatures below 0° C. in arctic waters, in the saturated saltenvironment of the Dead Sea, at pH values around 0 in coal deposits andgeothermal sulfur-rich springs, or at pH values greater than 11 insewage sludge. For example, several esterases and lipases cloned andexpressed from extremophilic organisms show high activity throughout awide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are in one aspect already ina vector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or in one aspect, the host cellcan be a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981) and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Oneaspect is to use cloning vectors, referred to as “fosmids” or bacterialartificial chromosome (BAC) vectors. These are derived from E. colif-factor which is able to stably integrate large segments of genomicDNA. When integrated with DNA from a mixed uncultured environmentalsample, this makes it possible to achieve large genomic fragments in theform of a stable “environmental DNA library.” Another type of vector foruse in the present invention is a cosmid vector. Cosmid vectors wereoriginally designed to clone and propagate large segments of genomicDNA. Cloning into cosmid vectors is described in detail in Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989). Once ligated into an appropriate vector, two ormore vectors containing different polyketide synthase gene clusters canbe introduced into a suitable host cell. Regions of partial sequencehomology shared by the gene clusters will promote processes which resultin sequence reorganization resulting in a hybrid gene cluster. The novelhybrid gene cluster can then be screened for enhanced activities notfound in the original gene clusters.

Therefore, in a one aspect, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, the at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides for enzymeactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors of activity, for antibodies that bind to a polypeptide ofthe invention, for nucleic acids that hybridize to a nucleic acid of theinvention, to screen for cells expressing a polypeptide of the inventionand the like. In addition to the array formats described in detail belowfor screening samples, alternative formats can also be used to practicethe methods of the invention. Such formats include, for example, massspectrometers, chromatographs, e.g., high-throughput HPLC and otherforms of liquid chromatography, and smaller formats, such as 1536-wellplates, 384-well plates and so on. High throughput screening apparatuscan be adapted and used to practice the methods of the invention, see,e.g., U.S. Patent Application No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a gene, e.g., a gene of the invention (a nucleic acid encoding apolypeptide of the invention). One or more, or, all the transcripts of acell can be measured by hybridization of a sample comprising transcriptsof the cell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention. These antibodiescan be used to isolate, identify or quantify a polypeptide of theinvention or related polypeptides. These antibodies can be used toisolate other polypeptides within the scope the invention or otherrelated polypeptides. The antibodies can be designed to bind to anactive site of a polypeptide of the invention. Thus, the inventionprovides methods of inhibiting enzymes using the antibodies of theinvention (see discussion above). The invention provides fragments ofthe enzymes of the invention, including immunogenic fragments of apolypeptide of the invention. The invention provides compositionscomprising a polypeptide or peptide of the invention and adjuvants orcarriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides of the invention or fragments comprising at least about5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutiveamino acids thereof, may also be used to generate antibodies which bindspecifically to the polypeptides or fragments. The resulting antibodiesmay be used in immunoaffinity chromatography procedures to isolate orpurify the polypeptide or to determine whether the polypeptide ispresent in a biological sample. In such procedures, a proteinpreparation, such as an extract, or a biological sample is contactedwith an antibody capable of specifically binding to one of thepolypeptides of the invention, or fragments comprising at least about 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutiveamino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention,or fragment thereof. After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention, or fragments comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 or more consecutive amino acids thereof canbe obtained by direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal, for example, a nonhuman.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides of the invention, or fragments comprising at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof. Alternatively, transgenic mice may be used toexpress humanized antibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention, orfragments comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 or more consecutive amino acids thereof may be used inscreening for similar polypeptides from other organisms and samples. Insuch techniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding. One such screening assay is described in “Methods forMeasuring Cellulase Activities”, Methods in Enzymology, Vol 160, pp.87-116.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., enzymes involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity) and/orantibodies of the invention. The kits also can contain instructionalmaterial teaching the methodologies and industrial uses of theinvention, as described herein. In one aspect, the kits are designed toaccommodate industrial scale levels of processing, e.g., of foods,feeds, oils and the like.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a modified chlorophyll catabolism pathway, or, a new ormodified enzyme (e.g., chlorophyllase) activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention, e.g., acoding sequence for an enzyme of the invention. See, e.g., WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using an enzyme of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

identity of all pathway substrates, products and intermediarymetabolites

identity of all the chemical reactions interconverting the pathwaymetabolites, the stoichiometry of the pathway reactions,

identity of all the enzymes catalyzing the reactions, the enzymereaction kinetics,

the regulatory interactions between pathway components, e.g. allostericinteractions, enzyme-enzyme interactions etc,

intracellular compartmentalization of enzymes or any othersupramolecular organization of the enzymes, and,

the presence of any concentration gradients of metabolites, enzymes oreffector molecules or diffusion barriers to their movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., amessage for a polypeptide involved in chlorophyll catabolism or havingan esterase (e.g., chlorophyllase) activity) or generating newtranscripts in a cell. This increased or decreased expression can betraced by testing for the presence of an enzyme of the invention or byenzyme activity assays. mRNA transcripts, or messages, also can bedetected and quantified by any method known in the art, including, e.g.,Northern blots, quantitative amplification reactions, hybridization toarrays, and the like. Quantitative amplification reactions include,e.g., quantitative PCR, including, e.g., quantitative reversetranscription polymerase chain reaction, or RT-PCR; quantitative realtime RT-PCR, or “real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001)Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., apolypeptide involved in chlorophyll catabolism or having an esterase(e.g., chlorophyllase) activity) or generating new polypeptides in acell. This increased or decreased expression can be traced bydetermining the amount of enzyme present or by activity assays.Polypeptides, peptides and amino acids also can be detected andquantified by any method known in the art, including, e.g., nuclearmagnetic resonance (NMR), spectrophotometry, radiography (proteinradiolabeling), electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, various immunological methods, e gimmunoprecipitation, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE),staining with antibodies, fluorescent activated cell sorter (FACS),pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry,Raman spectrometry, GC-MS, and LC-Electrospray andcap-LC-tandem-electrospray mass spectrometries, and the like. Novelbioactivities can also be screened using methods, or variations thereof,described in U.S. Pat. No. 6,057,103. Furthermore, as discussed below indetail, one or more, or, all the polypeptides of a cell can be measuredusing a protein array.

Enzymes

The invention provides novel compositions and methods for enzymaticallytreating, e.g., decoloring or “bleaching,” algal, animal (e.g., fish)and/or plant preparations, feeds, foods or oils comprising chlorophyll(the chlorophyll can be in the preparations, feeds, foods or oilsnaturally, as a contaminant, as an undesired composition in a processedproduct, etc). In one aspect, chlorophyll-containing orchlorophyll-contaminated compositions, e.g., algal, animal or plantpreparations, feeds, foods or oils are enzymatically treated using achlorophyllase or equivalent enzyme. Any polypeptide having an activitythat can modify a chlorophyll or chlorophyll metabolite, can be used ina composition or method of the invention.

Chlorophyllases

The polypeptides and/or peptides of the invention can have esteraseactivity, e.g., a chlorophyllase or a similar activity. The polypeptidesand/or peptides of the invention can include catalytic antibodies,enzymes, active sites, and the like. These polypeptides and/or peptidesof the invention having esterase (e.g., chlorophyllase) activity can beused in compositions or methods of the invention. For example, in oneaspect, compositions and methods of the invention enzymatically treatchlorophyll-containing or chlorophyll-contaminated compositions byhydrolyzing chlorophyll (FIG. 1A) to phytol (FIG. 1B) and chlorophyllide(FIG. 1C).

Any chlorophyllase, chlase or chlorophyll chlorophyllido-hydrolyase orpolypeptide having a similar activity (e.g., chlorophyll-chlorophyllidohydrolase 1 or chlase 1, or, chlorophyll-chlorophyllido hydrolase 2 orchlase 2, see, e.g., NCBI P59677_(—)1 and P59678, respectively) can beused in a composition or method of the invention. Any polypeptide (e.g.,enzyme or catalytic antibody) that catalyses the hydrolysis of achlorophyll ester bond to yield chlorophyllide and phytol can be used ina composition or method of the invention. Any isolated, recombinant orsynthetic or chimeric (a combination of synthetic and recombinant)polypeptide (e.g., enzyme or catalytic antibody) can be used, e.g., achlorophyllase, chlase or chlorophyll chlorophyllido-hydrolyase orpolypeptide having a similar activity can be used in a composition ormethod of the invention, see, e.g., Marchler-Bauer (2003) Nucleic AcidsRes. 31: 383-387.

In one aspect, the compositions and methods of the invention can bepracticed with enzymes as described in WO 0229022. For example, in oneaspect, the compositions and methods of the invention can compriserecombinant expression of enzymes, e.g., chlorophyllases, such aschlorophyllase-encoding polynucleotides. In one aspect, recombinantnucleic acid is expressed in whole cells, cell extracts or in vitro. Inone aspect, the enzyme-encoding polynucleotide is modified to result inproduction of altered levels of enzyme (e.g., chlorophyllase) in atransformed host cell.

In one aspect, the compositions and methods of the invention can bepracticed with known enzymes, such as chlorophyllases (including chlasesand chlorophyll chlorophyllido-hydrolyases) and related polypeptides arewell known in the art. For example, the Arabidopsis thalianachlorophyllase can be used as described, e.g., in NCBI entryNM_(—)123753 (where the enzyme having a sequence as set forth in SEQ IDNO:22 is encoded, e.g., by SEQ ID NO:21):

SEQ ID NO: 21AAAAAAAGTAAAGAAAAGAAAAACTAATAAAGAACAAAAAAAATGTCCTCTTCTTCATCAAGAAACGCCTTTGAAGATGGCAAATACAAATCAAATCTCTTAACCTTGGACTCATCATCTCGTTGCTGCAAAATAACACCGTCTTCTAGAGCTTCACCGTCTCCGCCAAAGCAGCTGTTGGTGGCTACGCCGGTGGAGGAAGGAGATTATCCGGTGGTGATGCTCCTCCATGGTTACCTTCTCTACAACTCCTTCTATTCTCAGCTTATGTTGCATGTCTCTTCTCATGGCTTCATCCTCATCGCTCCTCAGTTATATAGTATCGCCGGACCAGACACAATGGATGAGATTAAATCAACGGCGGAGATTATGGATTGGTTATCAGTAGGACTTAATCACTTTCTTCCAGCGCAAGTAACACCAAACCTATCCAAATTTGCCCTCTCCGGCCATAGCCGCGGTGGCAAAACCGCGTTTGCGGTCGCCTTAAAGAAATTTGGGTACTCCTCGAATCTAAAGATCTCGACATTGATCGGTATAGATCCAGTCGATGGAACAGGGAAAGGGAAACAAACCCCTCCTCCGGTGTTGGCTTACCTTCCAAACTCATTTGACCTAGACAAAACGCCTATACTTGTGATCGGTTCGGGGCTTGGTGAAACCGCTCGGAACCCATTATTCCCACCGTGTGCACCTCCCGGAGTGAATCACCGAGAGTTCTTTCGGGAATGTCAAGGTCCAGCATGGCATTTCGTTGCGAAGGATTATGGGCATTTGGACATGCTTGATGATGATACAAAAGGGATTAGAGGGAAGAGTTCTTATTGTTTGTGTAAGAATGGTGAAGAGAGGAGACCAATGAGGAGATTCGTTGGTGGACTTGTTGTATCATTTTTGAAGGCTTATTTGGAAGGAGATGATCGTGAATTAGTTAAGATCAAAGATGGGTGTCACGAGGATGTTCCCGTTGAAATTCAAGAGTTTGAGGTTATCATGTAAACATAAGTTTTTCTTTAGGGGCTGGTTTTTCTATTGTCAATATCATCAGCTTTTGTTGCTTATGGTTTTACAAACTTATATTGTACAACTCTTTAAGTCACCTCTTTGCTTATGATATTAACCCGATC SEQ ID NO: 22MSSSSSRNAFEDGKYKSNLLTLDSSSRCCKITPSSRASPSPPKQLLVATPVEEGDYPVVMLLHGYLLYNSFYSQLMLHVSSHGFILIAPQLYSIAGPDTMDEIKSTAEIMDWLSVGLNHFLPAQVTPNLSKFALSGHSRGGKTAFAVALKKFGYSSNLKISTLIGIDPVDGTGKGKQTPPPVLAYLPNSFDLDKTPILVIGSGLGETARNPLFPPCAPPGVNHREFFRECQGPAWHFVAKDYGHLDMLDDDTKGIRGKSSYCLCKNGEERRPMRRFVGGLVVSFLKAYLEGDDRELVKIKDGCHEDVPVEIQEFEVIM

The Ginkgo biloba chlorophyllase can be used as described, e.g., in NCBIentry AY292526:

SEQ ID NO: 23TTGAAAAACAAAAACGAAGAAGATGAACTCAGTACTTGCACACAGCCATCGGCCATGGTTTTAGTGAAGGATGTGTTCAGCGAAGGTCCTTTACCTGTTCAAATCCTCGCAATTCCACAAGCCAACTCATCTCCATGCTCAAAATTAGCAGACAAAAACGGAACTGCAACCACGCCTTCTCCTTGTCGGCCTCCTAAACCCCTGCTGATCGCTCTTCCTTCCCAACATGGAGATTATCCTCTCATCCTCTTTTTCCACGGCTATGTACTCCTCAATTCCTTCTATTCTCAACTCTTGCGCCATGTTGCTTCCCATGGATACATCGCCATAGCTCCTCAGATGTACAGTGTAATTGGCCCAAATACGACTCCAGAAATAGCCGATGCAGCGGCCATTACAGACTGGTTACGAGATGGACTCTCGGATAATCTTCCGCAAGCTTTAAACAATCATGTGAGGCCCAATTTTGAGAAATTTGTGCTAGCGGGGCACTCGCGCGGGGGTAAAGTGGCATTTGCACTTGCCCTAGGTCGAGTCTCGCAGCCATCTTTAAAGTACTCGGCCCTTGTAGGTCTTGATCCAGTCGATGGAATGGGAAAAGATCAACAAACCAGTCATCCTATTCTGTCATACAGAGAGCATTCCTTTGATTTGGGTATGCCAACATTAGTGGTAGGTTCGGGCCTGGGTCCGTGCAAAAGAAACCCTCTCTTCCCTCCCTGTGCTCCCCAAGGTGTTAACCACCATGATTTCTTCTACGAATGTGTCGCTCCTGCCTATCATTTTGTTGCCTCTGATTATGGGCATCTTGATTTCTTAGACGACGACACCAAAGGAATAAGAGGAAAGGCTACTTATTGCCTCTGTAAGAATGGGGAAGCAAGAGAGCCAATGCGGAAGTTTAGCGGTGGAATTGTGGTTGCATTTCTTCAAGCATTTCTTGGTGATAATCGTGGAGCCCTGAATGATATTATGGTTTATCCTTCACATGCTCCAGTCAAGATTGAGCCTCCAGAGTCTTTGGTTACAGAAGATGTAAAATCCCCAGAAGTCGAATTATTACGCCGGGCAGTTTGCAGATGATGTACCATGGTATTATGCATTAAAGGAATGTATTTGTTATTAAAAAAATATTAAGAAGTAAAAAAAAAAAAAAA SEQ ID NO: 24MVLVKDVFSEGPLPVQILAIPQANSSPCSKLADKNGTATTPSPCRPPKPLLIALPSQHGDYPLILFFHGYVLLNSFYSQLLRHVASHGYIAIAPQMYSVIGPNTTPEIADAAAITDWLRDGLSDNLPQALNNHVRPNFEKFVLAGHSRGGKVAFALALGRVSQPSLKYSALVGLDPVDGMGKDQQTSHPILSYREHSFDLGMPTLVVGSGLGPCKRNPLFPPCAPQGVNHHDFFYECVAPAYHFVASDYGHLDFLDDDTKGIRGKATYCLCKNGEAREPMRKFSGGIVVAFLQAFLGDNRGALNDIMVYPSHAPVKIEPPESLVTEDVKSPEVELLRRAVCR

The Brassica oleracea chlorophyllase can be used as described, e.g., inNCBI entry AF337546:

SEQ ID NO: 25ACACAAAAAAATATATAACACAAAGAAATAGAAGAAGGAAAAAATGTCCCCCTCCTTTCTTTTCTTTACTTTGTTTTTGATAAAGGAAATGTCCTCTTCATCATCAGCAAACTCCTTTGAGGACGGCAAATACAAAACAGATCTTTTAACAGTAGGCTTATCATCTTGCTGCTGGAAAAAGCCCTCCTCTTCTCCGACTCCGCAGTCTCCGCCGAAGAGGCTTTTGGTGGCAACGCCGGTGGAGGAAGGAGAATATCCGGTGGTGATGCTCCTCCATGGTTACCTTCTCTACAACTCATTTTATTCCCAGCTTATGTTGCATGTCTCTTCCCATGGCTTCATTGTCATCGCTCCGCAGTTATATAGCATTGCCGGACCAGACACCATGGATGAGATAAAATCAACGGCAGAGATTATTGATTGGTTATCGGTCGGACTAAACCACTTTCTTCCACCACAAGTAACACCAAACCTATCCAAGTTCGCACTCTCCGGCCATAGCCGTGGTGGGAAGACCGCATTTGCCTTGGCCTTAAAGAAATTTGGATACTCGTCCGACCTAAAGATCTCGGCATTGATAGGTATAGATGTTGGAACTGTTTTTTGGACAAATGGCTATGGCCAATATTCCGGTGAATTTTTCGAGCAATTTGATTGTCGAAATGACCGGATTGTGGAATCGTAGGATTCATTGTTATGAGCACTATGGTATAGTGTAATCATATATCAAAAACGAAGTTCGTTTGAATGAGAAATGAAAGTCTAAAATAGATTATTTGTAAAATATCTATATTAGAATTATGAGGTAAGAAACCTCTTGTGTTTAAAATGGAGAAGTTATAACAAAGTTATAAAAAACTTTGTAAACAATTTGGTGTGTTAGC SEQ ID NO: 26MSPSFLFFTLFLIKEMSSSSSANSFEDGKYKTDLLTVGLSSCCWKKPSSSPTPQSPPKRLLVATPVEEGEYPVVMLLHGYLLYNSFYSQLMLHVSSHGFIVIAPQLYSIAGPDTMDEIKSTAEIIDWLSVGLNHFLPPQVTPNLSKFALSGHSRGGKTAFALALKKFGYSSDLKISALIGIDVGTVFWTNGYGQYSGEFFEQFDCRNDRIVES

The Citrus sinensis chlorophyllase can be used as described, e.g., inNCBI entry Q9MV14:

SEQ ID NO: 27MAAMVDAKPAASVQGTPLLATATLPVFTRGIYSTKRITLETSSPSSPPPPKPLIIVTPAGKGTFNVILFLHGTSLSNKSYSKIFDHIASHGFIVVAPQLYTSIPPPSATNELNSAAEVAEWLPQGLQQNLPENTEANVSLVAVMGHSRGGQTAFALSLRYGFGAVIGLDPVAGTSKTTGLDPSILSFDSFDFSIPVTVIGTGLGGVARCITACAPEGANHEEFFNRCKNSSRAHFVATDYGHMDILDDNPSDVKSWALSKYFCKNGNESRDPMRRCVSGIVVAFLKDFFYGDAEDFRQILKDPSFAPIKLDSVEYIDASSMLTTTHVKV

Enzyme Preparations

Enzymes used in the methods of the invention can be formulated ormodified, e.g., chemically modified, e.g., to enhance oil solubility,stability, activity or for immobilization. For example, enzymes used inthe methods of the invention can be formulated to be amphipathic or morelipophilic. For example, enzymes used in the methods of the inventioncan be encapsulated, e.g., in liposomes or gels, e.g., alginatehydrogels or alginate beads or equivalents. Enzymes used in the methodsof the invention can be formulated in micellar systems, e.g., a ternarymicellar (TMS) or reverse micellar system (RMS) medium. Enzymes used inthe methods of the invention can be formulated as described in Yi (2002)J. of Molecular Catalysis B: Enzymatic, Vol. 19, No. 0, pgs 319-325. Forexample, amphipathic enzyme, e.g., chlorophyllase, in the form of aternary micellar (TMS) or reverse micellar system (RMS) medium can beencapsulated in alginate hydrogels. In one aspect, an enzyme, e.g., achlorophyllase, is prepared in aqueous buffer and retained in ahydrogel, e.g., TMS/alginate and RMS/alginate. One approach toencapsulating enzyme, e.g., chlorophyllase, can be emulsification and/orinternal gelation of the enzyme-TMS or -RMS system.

The enzymatic reactions of the methods of the invention can be done invitro, including, e.g. capillary arrays, as discussed below, or, inwhole cell systems. In one aspect, enzyme reactions of the methods ofthe invention are done in one reaction vessel or multiple vessels. Inone aspect, the enzymatic reactions of the methods of the invention aredone in a vegetable oil refining apparatus.

The compositions and methods of the invention can be practiced withimmobilized enzymes, e.g., immobilized chlorophyllase. The enzyme can beimmobilized on any organic or inorganic support. Exemplary inorganicsupports include alumina, celite, Dowex-1-chloride, glass beads andsilica gel. Exemplary organic supports include alginate hydrogels oralginate beads or equivalents.

In various aspects of the invention, immobilization of chlorophyllasecan be optimized by physical adsorption on various inorganic supports,including alumina, celite, Dowex-1-chloride, glass beads and silica gel.Enzymes used to practice the invention can be immobilized in differentmedia, including water, Tris-HCl buffer solution and a ternary micellarsystem containing Tris-HCl buffer solution, hexane and surfactant. Thehighest immobilization efficiency (84.56%) and specific activity (0.34mmol hydrolyzed chlorophyll mg protein-1 per min) were obtained whenchlorophyllase was suspended in Tris-HCl buffer solution and adsorbedonto silica gel.

Industrial and Medical Applications

The polypeptides, e.g., enzymes of the invention involved in chlorophyllcatabolism or having an esterase (e.g., chlorophyllase) activity, can beused in a variety of medical and industrial applications, as describedherein. The compositions and methods of the invention can be used inconjunction with any industrial use or pharmaceutical or medicalapplication for the treatment of chlorophyll-containing materials, e.g.,vegetable preparations, oil-comprising materials. For example, thecompositions and methods of the invention can be used with processes forconverting a non-hydratable phospholipid to a hydratable form, oildegumming, processing of oils from plants, fish, algae and the like, toname just a few applications. For example, the methods of the inventioncan be used with the processing of fats and oils as described, e.g., inJP Patent Application Publication H6-306386, describing convertingphospholipids present in the oils and fats into water-soluble substancescontaining phosphoric acid groups.

The compositions and methods of the invention can be used in conjunctionwith methods for processing plant oils, such as those derived from orisolated from rice bran, soy, canola, palm, cottonseed, corn, palmkernel, coconut, peanut, sesame, sunflower. The compositions and methodsof the invention can be used in conjunction with methods for processingessential oils, e.g., those from fruit seed oils, e.g., grapeseed,apricot, borage, etc. The compositions and methods of the invention canbe used in conjunction with methods for processing oils andphospholipids in different forms, including crude forms, degummed, gums,wash water, clay, silica, soapstock, and the like. The compositions andmethods of the invention can be used in conjunction with methods forprocessing high phosphorous oils (e.g., a soy bean oil), fish oils,animal oils, plant oils, algae oils and the like.

The compositions and methods of the invention can be used in conjunctionwith methods for processing and making edible oils, biodiesel oils,liposomes for pharmaceuticals and cosmetics, structured phospholipidsand structured lipids. The compositions and methods of the invention canbe used in conjunction with methods for oil extraction. The compositionsand methods of the invention can be used in conjunction with methods formaking various soaps.

The methods can further comprise modifying pH (e.g., increasing pH) topromote aqueous separation of chlorophyllide. Thus, the compositions andmethods of the invention can also comprise a caustic neutralizationprocesses, e.g., with caustic-neutralized pH conditions. In one aspect,the compositions and methods of the invention comprise a neutralizationstep, e.g., in treating “chemically refined oils”, e.g., usingchlorophyllases and/or in the separation chlorophyllide. Thecompositions and methods of the invention can comprise modifying pH topromote aqueous separation of chlorophyllide.

In one aspect, the compositions and methods of the invention compriseuse of adsorbent-free or reduced adsorbent silica refining devices andprocesses, which are known in the art, e.g., using TriSyl SilicaRefining Processes (Grace Davison, Columbia, Md.), or, SORBSIL R™silicas (INEOS Silicas, Joliet, Ill.).

Enzymatic Treatment, or “Bleaching” or Decoloring Processes

The invention provides novel compositions and methods for enzymaticallytreating, e.g., decoloring or “bleaching,” algal, animal (e.g., fish)and/or plant preparations, feeds, foods or oils, as illustrated in FIGS.8 to 16. In one aspect, chlorophyll-containing orchlorophyll-contaminated foods or oils are treated. For example, inalternative aspects, vegetable oils, including oils processed fromoilseeds, such as canola (rapeseed) oil or soybean oil, or oil fruits,such as palm oil, are processes using the compositions and/or methods ofthe invention.

At least one step in this exemplary method involves use of an enzyme,e.g., a chlorophyllase enzyme that can hydrolyze chlorophyll to phytoland chlorophyllide. In alternative aspects, one, several or all stepsuse an enzyme. The reaction can be in vitro or in vivo.

FIG. 8 illustrates the reaction of an exemplary esterase of theinvention in chlorophyll degradation—the chlorophyllase (chlase)catalyzes hydrolysis of an ester bond in chlorophyll to yieldchlorophyllide and phytol, where the chlorophyllide enters the aqueousphase due to a hydrophilic porphyrin ring, and the phytol separates intoan oil (hydrophobic) phase. In one aspect of a process of the inventionthe hydrophilic porphyrin ring is separated with gum/water fractionusing any one of the many well-known methods.

FIG. 9 illustrates and compares traditional versus an exemplaryenzymatic decoloring (bleaching) process of the invention, where theenzymatic bleaching process can incorporate an esterase of theinvention. In the traditional method crude vegetable oil is degummed,(optionally, caustic neutralized), bleached using, e.g., clay adsorptionwith subsequent clay disposal, and deodorization to produce “refined,bleached and deodorized” or RBD oil. In this exemplary enzymaticbleaching process of the invention, the crude vegetable oil is degummed,(optionally, caustic neutralized), bleached using, e.g., a polypeptideof the invention, such as a chlorophyllase of the invention, withsubsequent aqueous separation of the chlorophyllide, followed bydeodorization to produce a “refined, bleached and deodorized” or RBDoil. The need for the degumming depends on phosphorus content and otherfactors (all known in the art). Soy and canola are typically degummed.

FIG. 10 illustrates an exemplary enzymatic decoloring (bleaching)process of the invention—a combined degumming-bleaching (“decoloring”)process. In this exemplary enzymatic bleaching process of the invention,the crude vegetable oil is degummed and enzymatically bleached using apolypeptide of the invention, such as an esterase, e.g., achlorophyllase, of the invention in one step, or “one pot.” Thedegumming can be a “traditional” or an enzymatic degumming, e.g.,involving phospholipid(s) and/or hydrolysis. In one aspect, theexemplary process of the invention comprises a subsequent aqueousseparation step to remove the reaction product chlorophyllide, gumand/or soap. In one aspect, this is followed by deodorization to producea “refined, bleached and deodorized” or RBD oil.

FIG. 11 illustrates an exemplary enzymatic decoloring (bleaching)process of the invention that combines degumming, enzymatic bleaching(“decoloring”) and caustic neutralization steps. In this exemplaryenzymatic bleaching process of the invention, the crude vegetable oil isdegummed, neutralized and enzymatically bleached using a polypeptide ofthe invention, such as an esterase, e.g., a chlorophyllase, of theinvention in one step, or “one pot.” The degumming can be a“traditional” or an enzymatic degumming, e.g., involving phospholipid(s)and/or hydrolysis. In one aspect, the exemplary process of the inventioncomprises a subsequent aqueous separation step to remove the reactionproduct chlorophyllide, gum and/or soap.

FIG. 12 illustrates an exemplary enzymatic decoloring (bleaching)process of the invention that comprises application of a polypeptide ofthe invention, such as an esterase, e.g., a chlorophyllase to an oilseedpreparation, followed by a subsequent aqueous separation step (toremove, e.g., the reaction product chlorophyllide, or gums and/orsoaps), followed by the processes illustrated in FIG. 9, 10, or 11.

FIG. 13 illustrates a general oilseed refining scheme comprisingextraction, refining and modification of an oilseed, where in additionto a polypeptide of the invention, such as an esterase, e.g., achlorophyllase to an oilseed in one or several or all of these steps,other polypeptides and/or chemicals are also added, e.g., cellulase,hemicellulase, protease, pectinase, phospholipase A, B, C and/or D,esterase (e.g., a selective esterase), a lipase (e.g., 1,3 lipase), aselective lipase, a known chlorophyllase or other enzyme involved inchlorophyll catabolism, and the like.

FIG. 14 illustrates an exemplary industrial process of the invention—abiodegumming process, comprising use of a phospholipase A and at leastone polypeptide of the invention having chlorophyllase enzyme activity.The at least one polypeptide of the invention having chlorophyllaseactivity can be added to one or several or all of the following steps:added to the crude oil, in the degumming process or in the degummed oil,a storage or holding tank, with the phospholipase A (e.g., in “the daytank” of the figure) and/or the caustic tank.

FIG. 15 illustrates another exemplary industrial process of theinvention comprising use of at least one polypeptide of the inventionhaving chlorophyllase enzyme activity. The at least one polypeptide ofthe invention having chlorophyllase activity can be added to one orseveral or all of the following steps: added to the crude oil, in thedegumming process or in the degummed oil, a storage or holding tank, acaustic tank and/or a retention mixer.

FIG. 16 illustrates another exemplary industrial process of theinvention comprising use of at least one polypeptide of the inventionhaving chlorophyllase enzyme activity. In this exemplary process,phospholipase C (PLC) is added into the degumming process or in thedegummed oil with the chlorophyllase enzyme of the invention. The atleast one polypeptide of the invention having chlorophyllase activitycan be added to one or several or all of the following steps: added tothe crude oil, in the degumming process or in the degummed oil (with aPLC), a storage or holding tank, a caustic tank and/or a retentionmixer.

Oil Degumming and Vegetable Oil Processing

The compositions and methods of the invention can be used in variousvegetable oil processing steps, such as in vegetable oil extraction,particularly, in the removal of “phospholipid gums” in a process called“oil degumming,”.

The compositions and methods of the invention can be used in methods forprocessing vegetable oils from various sources, such as rice bran,soybeans, rapeseed, peanuts and other nuts, sesame, sunflower, palm andcorn. The methods can used in conjunction with processes based onextraction with as hexane, with subsequent refining of the crudeextracts to edible oils. The first step in the refining sequence is theso-called “degumming” process, which serves to separate phosphatides bythe addition of water. The material precipitated by degumming isseparated and further processed to mixtures of lecithins. The commerciallecithins, such as soybean lecithin and sunflower lecithin, aresemi-solid or very viscous materials. They consist of a mixture of polarlipids, mainly phospholipids, and oil, mainly triglycerides. Thecompositions and methods of the invention can be used before or afterany step in a process, or before or after any combination of steps, orbefore or after all of the steps, in a process, e.g., prior to, duringor following mechanical and/or chemical extraction, degumming and/orbleaching and the like.

The compositions and methods of the invention can be used in (i.e., inconjunction with) any “degumming” procedure, including water degumming,ALCON oil degumming (e.g., for soybeans), safinco degumming, “superdegumming,” UF degumming, TOP degumming, uni-degumming, dry degummingand ENZYMAX™ degumming See, e.g., U.S. Pat. Nos. 6,355,693; 6,162,623;6,103,505; 6,001,640; 5,558,781; 5,264,367. Compositions and methods ofthe invention can be used in any oil processing method, e.g., degummingor equivalent processes. For example, compositions and methods of theinvention can be used in processes as described in U.S. Pat. Nos.5,558,781; 5,288,619; 5,264,367; 6,001,640; 6,376,689; WO 0229022; oildegumming as described, e.g., in WO 98/18912; processes as described inJP Application No.: H5-132283 (filed Apr. 25, 1993); EP Applicationnumber: 82870032.8, and the like. Various “degumming” proceduresincorporated by the methods of the invention are described in Bockisch,M. (1998) In Fats and Oils Handbook, The extraction of Vegetable Oils(Chapter 5), 345-445, AOCS Press, Champaign, Ill. The compositions andmethods of the invention can be used in the industrial application ofenzymatic degumming of triglyceride oils as described, e.g., in EP 513709.

In one aspect, compositions and methods of the invention are used totreat vegetable oils, e.g., crude oils, such as rice bran, soy, canola,flower and the like. In one aspect, this improves the efficiency of thedegumming process. In one aspect the methods of the invention result inthe improved separation of chlorophyll from the oil phase, e.g., duringcentrifugation. The improved separation of these phases can result inmore efficient removal of chlorophylls from the oil, including bothhydratable and nonhydratable oils.

The compositions and methods of the invention can be used in theindustrial application of enzymatic degumming as described, e.g., in CA1102795, which describes a method of isolating polar lipids from cereallipids by the addition of at least 50% by weight of water. This methodis a modified degumming in the sense that it utilizes the principle ofadding water to a crude oil mixture.

In one aspect, the invention provides enzymatic processes comprising useof compositions and methods of the invention comprising hydrolysis ofhydrated phospholipids in oil at a temperature of about 20° C. to 40°C., at an alkaline pH, e.g., a pH of about pH 8 to pH 10, using areaction time of about 3 to 10 minutes.

In various exemplary processes of the invention, a number of distinctsteps comprise the degumming process preceding the core bleaching anddeodorization refining processes. These steps include heating, mixing,holding, separating and drying. Following the heating step, water andoften acid are added and mixed to allow the insoluble phospholipid “gum”to agglomerate into particles which may be separated. While waterseparates many of the phosphatides in degumming, portions of thephospholipids are non-hydratable phosphatides (NHPs) present as calciumor magnesium salts. Degumming processes address these NHPs by theaddition of acid. Following the hydration of phospholipids, the oil ismixed, held and separated by centrifugation. Finally, the oil is driedand stored, shipped or refined. The resulting gums are either processedfurther for lecithin products or added back into the meal. As notedabove, the compositions and methods of the invention can be used beforeor after any of these steps, or before or after any combination ofsteps, or before or after all of the steps, in any processing method.

Upon completion of an enzyme treatment of the invention, the treatedliquid (e.g., oil) is separated with an appropriate means such as acentrifugal separator and the processed oil is obtained. In one aspect,compounds produced by enzyme modification of chlorophyll are partiallyor completely transferred into the aqueous phase and removed from theoil phase. Upon completion of the enzyme treatment, if necessary, theprocessed oil can be additionally washed with water or organic orinorganic acid such as, e.g., acetic acid, phosphoric acid, succinicacid, and the like, or with salt solutions.

In one exemplary process for ultra-filtration degumming, an enzyme usedin a method of the invention is bound to a filter or the enzyme is addedto an oil prior to filtration. Enzymes used in compositions or methodsof the invention can be immobilized to any substrate, e.g., filters,fibers, columns, beads, colloids, gels, hydrogels, meshes and the like.

Compositions and methods of the invention can be used to improve oilextraction, oil degumming and caustic neutralization (e.g., vegetableoils). In one aspect, a composition or method of the invention and atleast one plant cell wall degrader (e.g., a cellulase, a hemicellulaseor the like, to soften walls and increase yield at extraction) is usedin a process of the invention. In an exemplary method, to improve oilextraction and oil degumming, a phospholipase, e.g., a phospholipase C,or another hydrolase (e.g., a cellulase, a hemicellulase, an esterase, aprotease and/or a phosphatase) is used. For example, in one aspect,during a crushing step associated with oil production (including but notlimited to soybean, canola, sunflower, rice bran oil) a phospholipase orother enzyme can be used. By using enzymes prior to or in place ofsolvent extraction, it is possible to increase oil yield and reduce theamount of hydratable and non-hydratable phospholipids in the crude oil.The overall reduction of phospholipids in the crude oil will result inimproved yields during refining with the potential for eliminating therequirement for a separate degumming step prior to bleaching anddeodorization.

Compositions and methods of the invention also can be practiced usingprocesses as described in U.S. Pat. No. 5,414,100. For example, in oneaspect, the methods or compositions further comprise chromatographicprocesses for deacidification of vegetable oils at ambient temperature.These processes can be retrofitted into deacidification operations usingmiscella refining or solvent extraction, crude vegetable oil isdissolved in a solvent such as isopropyl alcohol and passed through acolumn of activated alumina (aluminum oxide) at room temperature. Theprocess, which eliminates physical contact between both oil and analkaline reagent and oil and water, simplifies subsequent bleachingprocesses by also removing some color pigments. The spent alumina can bereactivated by washing it with a dilute solution of sodium hydroxide orpotassium hydroxide.

Compositions and methods of the invention also can be practiced usingprocesses as described in JP57156482, 1982 (application no.JP19810040794 19810320), describing refining vegetable fats or oils asby-products.

Compositions and methods of the invention also can be practiced usingprocesses as described in U.S. Pat. No. 5,315,021. For example, in oneaspect, the methods or compositions of the invention can be practicedwith processes for removing chlorophyll color impurities from vegetableoils. The processes can comprise dispersing a source of phosphoric acidin vegetable oil to form a mixture having a moisture content of lessthan 0.1% by weight which mixture is maintained at a temperature in therange of 70° C. to 160° C. until a precipitate containing chlorophyllcolor impurities is formed. This can be followed separating theprecipitated material from the oil to remove the chlorophyll colorimpurities with the precipitated material, e.g., during conventional oilprocessing up to and including the removal of bleaching clay from theoil.

Enzymatic Processing of Oilseeds

The compositions and methods of the invention can be used for enzymaticprocessing of oilseeds, including soybean, canola (rapeseed), coconut,avocado and olive paste. In one aspect, these processes of the inventioncan increase the oil yield and to improve the nutritional quality of theobtained meals. In some aspects, enzymatic processing of oilseeds usingthe enzymes and methods of the invention will provide economical andenvironmental benefits, as well as alternative technologies for oilextraction and processing food for human and animal consumption. Inalternative aspects, the processes of the invention further comprise useof phospholipases, proteases, phosphatases, phytases, xylanases,amylases (e.g., α-amylases), glucanases (e.g., 13-glucanases),polygalacturonases, galactolipases, cellulases, hemicellulases,pectinases and other plant cell wall degrading enzymes, as well as mixedenzyme preparations and cell lysates. In alternative aspects, theprocesses of the invention can be practiced in conjunction with otherprocesses, e.g., enzymatic treatments, e.g., with carbohydrases,including cellulase, hemicellulase and other side degrading activities,or, chemical processes, e.g., hexane extraction of soybean oil. Theenzymatic treatment can increase the oil extractability by 8-10% whenthe enzymatic treatment is carried out prior to the solvent extraction.

In alternative aspects, the processes of the invention can be practicedwith aqueous extraction processes. The aqueous extraction methods can beenvironmentally cleaner alternative technologies for oil extraction. Theprocesses of the invention can also use enzymes that hydrolyze thestructural polysaccharides forming the cell wall of oilseeds, or thathydrolyze the proteins which form the cell and lipid body membranes,e.g., utilizing digestions comprising cellulase, hemicellulase, and/orprotopectinase for extraction of oil from soybean cells. In one aspect,methods are practiced with an enzyme of the invention as described byKasai (2003) J. Agric. Food Chem. 51:6217-6222, who reported that themost effective enzyme to digest the cell wall was cellulase.

In one aspect, proteases are used in combination with the methods of theinvention. The combined effect of operational variables and enzymeactivity of protease and cellulase on oil and protein extraction yieldscombined with other process parameters, such as enzyme concentration,time of hydrolysis, particle size and solid-to-liquid ratio has beenevaluated. In one aspect, methods of the invention are practiced withprotocols as described by Rosenthal (2001) Enzyme and Microb. Tech.28:499-509, who reported that use of protease can result insignificantly higher yields of oil and protein over the control whenheat treated flour is used.

In one aspect, complete protein, pectin, and hemicellulose extractionare used in combination with the methods of the invention. The plantcell consists of a series of polysaccharides often associated with orreplaced by proteins or phenolic compounds. Most of these carbohydratesare only partially digested or poorly utilized by the digestive enzymes.The disruption of these structures through processing or degradingenzymes can improve their nutrient availability. In one aspect, methodsof the invention are practiced with protocols as described by Ouhida(2002) J. Agric. Food Chem. 50:1933-1938, who reported that asignificant degradation of the soybean cell wall cellulose (up to 20%)has been achieved after complete protein, pectin, and hemicelluloseextraction.

In one aspect, the methods of the invention further compriseincorporation of various enzymatic treatments in the treatment of seeds,e.g., canola seeds, these treatments comprising use of proteases,cellulases, and hemicellulases (in various combinations with each otherand with one or more enzymes of the invention). For example, the methodscan comprise enzymatic treatments of canola seeds at 20 to 40 moistureduring the incubation with enzymes prior to a conventional process; asdescribed, e.g., by Sosulski (1990) Proc. Can. Inst. Food Sci. Technol.3:656. The methods of the invention can further comprise incorporationof proteases, α-amylases, polygalacturonases (in various combinationswith each other and with one or more enzymes of the invention) tohydrolyze cellular material in coconut meal and release the coconut oil,which can be recovered by centrifugation, as described, e.g., by McGlone(1986) J. of Food Sci. 51:695-697. The methods of the invention canfurther comprise incorporation of pectinases, α-amylases, proteases,cellulases in different combinations (with each other and with one ormore enzymes of the invention) to result in significant yieldimprovement (˜70% in the best case) during enzymatic extraction ofavocado oil, as described, e.g., by Buenrostro (1986) Biotech. Letters8(7):505-506. In processes of the invention for olive oil extraction,olive paste is treated with cellulase, hemicellulase, poligalacturonase,pectin-methyltransferase, protease and their combinations (with eachother and with one or more enzymes of the invention), as described,e.g., by Montedoro (1976) Acta Vitamin. Enzymol. (Milano) 30:13.

In one aspect, the methods of the invention further compriseincorporation of various enzymatic treatments in the treatment of seeds,e.g., canola seeds, these treatments comprising use of proteases,cellulases, and hemicellulases (in various combinations with each otherand with one or more enzymes of the invention). For example, the methodscan comprise enzymatic treatments of canola seeds at 20 to 40 moistureduring the incubation with enzymes prior to a conventional process; asdescribed, e.g., by Sosulski (1990) Proc. Can. Inst. Food Sci. Technol.3:656. The methods of the invention can further comprise incorporationof proteases, α-amylases, polygalacturonases (in various combinationswith each other and with one or more enzymes of the invention) tohydrolyze cellular material in coconut meal and release the coconut oil,which can be recovered by centrifugation, as described, e.g., by McGlone(1986) J. of Food Sci. 51:695-697. The methods of the invention canfurther comprise incorporation of pectinases, α-amylases, proteases,cellulases in different combinations (with each other and with one ormore enzymes of the invention) to result in significant yieldimprovement (˜70% in the best case) during enzymatic extraction ofavocado oil, as described, e.g., by Buenrostro (1986) Biotech. Letters8(7):505-506. In processes of the invention for olive oil extraction,olive paste is treated with cellulase, hemicellulase, poligalacturonase,pectin-methyltransferase, protease and their combinations (with eachother and with one or more enzymes of the invention), as described,e.g., by Montedoro (1976) Acta Vitamin. Enzymol. (Milano) 30:13.

In one aspect, the compositions and methods of the invention can bepracticed with methods as described in U.S. Pat. No. 6,376,689. Forexample, in one aspect, the compositions and methods of the inventioncan comprise a single-step acid degumming/decolorizing process thatremoves chlorophyll-type compounds from vegetable oils from seeds,especially frost damaged seeds which have large amounts ofchlorophyll-type compounds. In one aspect, the methods of the inventionfurther comprise a mixture of aqueous sulfuric and phosphoric acids thatis blended with the oil to remove chlorophyll-type compounds from theoil. The purified oil can have less than about 5 ppm chlorophyll-typecompounds, less than about 50 ppm phosphorus or less than about 1.0weight percent free fatty acids.

Purification of Phytosterols from Vegetable Oils

The compositions (e.g., esterases) and methods of the invention can alsobe used in conjunction with methods and processes for the purificationof phytosterols and triterpenes, or plant sterols, from vegetable oils.Phytosterols that can be purified using methods of the invention includeβ-sitosterol, campesterol, stigmasterol, stigmastanol, β-sitostanol,sitostanol, desmosterol, chalinasterol, poriferasterol, clionasterol andbrassicasterol. Plant sterols are important agricultural products forhealth and nutritional industries. Thus, compositions (e.g., esterases)and methods of the invention can be used to make emulsifiers forcosmetic manufacturers and steroidal intermediates and precursors forthe production of hormone pharmaceuticals. The compositions (e.g.,esterases) and methods of the invention can be used to make (e.g.,purify) analogs of phytosterols and their esters for use ascholesterol-lowering agents with cardiologic health benefits. Thecompositions (e.g., esterases) and methods of the invention can be usedto purify plant sterols to reduce serum cholesterol levels by inhibitingcholesterol absorption in the intestinal lumen. The compositions (e.g.,esterases) and methods of the invention can be used to purify plantsterols that have immunomodulating properties at extremely lowconcentrations, including enhanced cellular response of T lymphocytesand cytotoxic ability of natural killer cells against a cancer cellline. The compositions (e.g., esterases) and methods of the inventioncan be used to purify plant sterols for the treatment of pulmonarytuberculosis, rheumatoid arthritis, management of HIV-infested patientsand inhibition of immune stress, e.g., in marathon runners.

The compositions (e.g., esterases) and methods of the invention can beused to purify sterol components present in the sterol fractions ofcommodity vegetable oils (e.g., coconut, canola, cocoa butter, corn,cottonseed, linseed, olive, palm, peanut, rice bran, safflower, sesame,soybean, sunflower oils), such as sitosterol (40.2-92.3%), campesterol(2.6-38.6%), stigmasterol (0-31%) and 5-avenasterol (1.5-29%).

Vegetable Oil Refining Apparatus

The invention provides product of manufacture comprising a degummingsystem for the enzymatic treatment of chlorophyll-containing orchlorophyll-contaminated compositions comprising (a) a vegetable oilrefining apparatus; and (b) a polypeptide having an chlorophyllaseactivity operably integrated into the vegetable oil refining apparatus,wherein the activity of the polypeptide comprises catalysis of achlorophyll-modifying reaction, and the vegetable oil refining apparatuscan react a chlorophyll-containing or chlorophyll-contaminatedcomposition with the polypeptide to under conditions wherein thepolypeptide can catalyze a chlorophyll-modifying reaction.

The products of manufacture of the invention can comprise any vegetableoil refining apparatus or combination thereof, e.g., an oil leavingexpellor (e.g., from Pennwalt Corp.), or a gravitational gum separationdevice.

The invention provides product of manufacture comprising immobilizedenzymes, e.g., an immobilized chlorophyllase, e.g., an esterase of theinvention. In one aspect of the product of manufacture, thechlorophyllase comprises a silica-immobilized chlorophyllase. The silicacomprises a silica gel or equivalent. The silica comprises a TriSylSilica or a SORBSIL R™ silica.

In one aspect, the products of manufacture of the invention compriseapparatus for adjusting pH, e.g., increasing pH (“caustic treatment”),and then, alternatively, neutralizing pH.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Exemplary Esterase Activity Assay

The following example demonstrates an exemplary esterase (chlorophyllaseactivity) assay for isolating and characterizing enzymes of theinvention and the nucleic acids that encode them, and to determine if apolypeptide is within the scope of the invention.

Esterases were screened for activity on chlorophyll from spinach toproduce chlorophyllide. In this exemplary esterase (chlorophyllaseactivity) assay the esterase screening format comprises:

-   -   Plates screened in duplicate.    -   Positive (CHLase) & negative controls on each plate.    -   1 mM CHL, 20% cell lysate, 20% acetone, pH 7.5, 0.01% HBT.    -   24 hr incubation time at 30° C. in the dark.    -   100 mL reaction volume.    -   Analysis by LC-VIS; injection of 1 mL sample.

This esterase screening method used HPLC to analyze reaction products.FIG. 2 and FIG. 3 illustrate data showing the results of the esterase(chlorophyllase activity) activity assay using the indicated exemplaryenzymes of the invention.

-   -   For the HPLC:    -   Column: Cromolith SpeedROD RP-18e 50-4, 6 mm (Cat# UM1082/086)    -   Flow: 1.0 mL/min; Injection: 1.0 mL.

T (min) A B C 0 10% 80% 10% 2.3 10% 80% 10% 2.31  0% 50% 50% 4  0% 50%50% 4.1 10% 80% 10% 7 10% 80% 10% A: H2O B: MEOH + 1 mM NH4Oac C: MTBE

DAD signal L (nm) Bw Reference 1 Bw 1 660 20 710 nm 10 Compound Rt CHLa4.20 CHLb 4.15 PHPa 4.30 PHPb 4.25 CHPa 0.85 CHPb 0.80 PHBa 1.00 PHBb0.95

The data illustrated in FIG. 2 illustrates increased levels of reactionproduct between 24 hr and 48 hr time points, were the levels of reactionproduct indicate chlorophyllase activity for SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18 and SEQ ID NO:20. The data illustrated in FIG. 3 illustratesincreased levels of reaction product between 24 hr and 48 hr timepoints, were the levels of reaction product indicate chlorophyllaseactivity for SEQ ID NO:10.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of enzymatic treatment comprising: a) providing at least one polypeptide according to the polypeptide of SEQ ID NO: 22 or the polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 21 and having a chlorophyllase activity b) de-coloring a pheophytin-containing composition under conditions wherein the at least one polypeptide catalyzes hydrolysis of pheophytin to generate pheophorbide and a phytol; and c) separating said pheophorbide and phytol.
 2. The method of claim 1, wherein the polypeptide is immobilized on: an array, a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, a capillary tube, an inorganic support or organic support, and optionally the support comprises: an alumina, celite, Dowex-1-chloride, glass beads, silica, silica gel, alginate hydrogel, or alginate bead.
 3. The method of claim 1, wherein the pheophytin containing composition: (a) is derived from a plant, an animal or an algae, or a mixture thereof, (b) comprises a plant material, plant oil or plant extract, (c) comprises a vegetable oil or a seed oil, (d) comprises a palm oil or a canola oil, (e) comprises a crude oil or a refined oil, (f) comprises an undiluted crude oil preparation, or (g) comprises an algae preparation.
 4. The method of claim 1, wherein at least one step of the treatment is performed in a reaction vessel, and optionally the reaction vessel comprises a gravitational gum separation device or a holding tank.
 5. The method of claim 1, wherein at least one step of the treatment is performed in a cell extract or in a whole cell.
 6. The method of claim 1, wherein the treatment further comprises modifying pH to promote aqueous separation of a pheophorbide.
 7. The method of claim 1, wherein the treatment is combined with a caustic neutralization step.
 8. The method of claim 1, wherein polypeptide is used with a phospholipase and optionally the phospholipase is a phospholipase C.
 9. The method of claim 1, wherein the treatment is included in a degumming process.
 10. The method of claim 1, wherein the treatment further comprises the removal of residual chlorophyll or pheophytin; pesticides; or a polycyclic aromatic hydrocarbons.
 11. The method of claim 1, wherein the enzymatic treatment of a pheophtin-containing composition is combined with a degumming process and a caustic neutralization step. 